Polynucleotide encoding a novel cysteine protease of the calpain superfamily, CAN-12, and variants thereof

ABSTRACT

The present invention provides novel polynucleotides encoding CAN-12 polypeptides, fragments and homologues thereof. The present invention also provides polynucleotides encoding variants of CAN-12 polypeptides, CAN-12v1 and CAN-12v2. Also provided are vectors, host cells, antibodies, and recombinant and synthetic methods for producing said polypeptides. The invention further relates to diagnostic and therapeutic methods for applying these novel CAN-12, CAN-12v1, and CAN-12v2 polypeptides to the diagnosis, treatment, and/or prevention of various diseases and/or disorders related to these polypeptides, particularly neuro- and musculo-degenerative conditions. The invention further relates to screening methods for identifying agonists and antagonists of the polynucleotides and polypeptides of the present invention.

This application claims benefit to provisional application U.S. Ser. No.60/281,253 filed Apr. 3, 2001; to provisional application U.S. Ser. No.60/288,768, filed May 4, 2001; to provisional application U.S. Ser. No.60/296,180, filed Jun. 6, 2001; to provisional application U.S. Ser. No.60/300,620, filed Jun. 25, 2001. The entire teachings of the referencedapplications are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention provides novel polynucleotides encoding CAN-12polypeptides, fragments and homologues thereof. The present inventionalso provides polynucleotides encoding variants of CAN-12 polypeptides,CAN-12v1 and CAN-12v2. Also provided are vectors, host cells,antibodies, and recombinant and synthetic methods for producing saidpolypeptides. The invention further relates to diagnostic andtherapeutic methods for applying these novel CAN-12, CAN-12v1, andCAN-12v2 polypeptides to the diagnosis, treatment, and/or prevention ofvarious diseases and/or disorders related to these polypeptides,particularly neuro- and musculo-degenerative conditions. The inventionfurther relates to screening methods for identifying agonists andantagonists of the polynucleotides and polypeptides of the presentinvention.

BACKGROUND OF THE INVENTION

Cysteine or thiol proteases contain a reactive sulphydral moietyactivated by an adjacent histidine. Hydrolysis of the substrates peptidebond is initiated when the cysteine sulfur attacks the carbon in thepeptide bond forming a thiol-enzyme intermediate, liberating the aminoportion of the peptide. The thiol-enzyme intermediate is hydrolyzed bywater releasing the substrates C-terminus and restoring the enzyme.There are over 20 some families of cysteine proteases. [Rawlings N. D.,& Barrett A. J. Families of cysteine peptidases. Methods in Enzymol. 244461-486 (1994)]. The present invention relates to a thiol protease ofthe C2 family that includes the calpain superfamily.

Calpains are calcium-activated intracellular neutral cysteine proteases(EC 3.4.22.17) (for reviews see Sorimachi et al., Structure andphysiological function of calpains. Biochem J. 328:721-32, 1997;Carafoli E and Molinari M. Calpain: a protease in search of a function?Biochem Biophys Res Commun 247:193-203, 1998). Some calpains areexpressed ubiquitously while others are tissue-specific. μ-Calpain andm-calpains appear in all tissues, p94 is skeletal muscle specific whilenCL-2 is stomach specific. (Sorimachi et al., Structure andphysiological function of calpains. Biochem J. 328:721-32, 1997). Thebest characterized are μ-calpain and m-calpains which consist of twosubunits. An 80 kDa large subunit contains both Ca²⁺ binding sites andthe catalytic activity and small 30 kDa subunit with a separate set ofCa²⁺ binding sites. All the proteolytic activity is contained in thelarger subunit of both μ- and m-calpain. In the presence of PEG orchaperones the large subunit is catalytically activated in the absenceof the smaller subunit. Other calpains, for example nCL-2 and p94, areproteolytically active monomers with homology to the μ-calpain andm-calpains large subunit.

The large (catalytic) subunit has four domains (I-IV)(Hosfield et al.,Crystal structure of calpain reveals the structural basis forCa(2+)-dependent protease activity and a novel mode of enzymeactivation. EMBO J 18:6880-9, 1999; Strobl et al., The crystal structureof calcium-free human m-calpain suggests an electrostatic switchmechanism for activation by calcium. Proc Natl Acad Sci USA. 97:588-92,2000). The N-terminus (domain I) contains an alpha helical region and asite of autocatalytic cleavage. Domain II contains the catalyticallyactive domain with the active site amino acids (m-calpain residuesCys105, His262, & Asn286). Domain III contains the linker between theCa²⁺ binding domain (in domain IV) and links Ca²⁺ binding to proteolyticactivity. Domain IV contains a calmodulin-like Ca²⁺ binding regions withEF hands. p94 (also called calpain 3) is similarly organized withdomains I-IV, but, also contains a proline-rich N-terminus and twounique insertion loops (IS1 and IS2). nCL-2 is also active as a largemonomer with domains I-IV; however, a splice variant (nCL-2′) lacksdomains III & IV, but maintains proteolytic activity.

Calpains are responsible for limited intracellular proteolytic cleavage,as opposed to complete proteolytic digestion. The proteolysis modifiesprotein function both specifically and irreversibly. Numerous proteinshave been identified as calpain substrates (Carafoli E and Molinari MCalpain: a protease in search of a function? Biochem Biophys Res Commun247:193-203, 1998; Hayes et al., Drug News Perspect 11:215-222, 1998).The best-characterized substrates are large cytostructural and/ormembrane associated proteins, calmodulin-binding proteins andtranscriptional factors. Physiologically significant substrates forcalpain include kinases, phosphatases, channel proteins and cytoskeletalproteins that link transmembrane receptors to the membrane skeleton.Proteolytic modification of these proteins may have fundamental roles indevelopment, differentiation, and cellular transformation in response tocell signaling, cell-cell and/or cell-extracellular matrix interactions.In platelets, calpain activation appears to be linked to clustering ofthe integrin receptor aIIb3 (Fox J E On the role of calpain and Rhoproteins in regulating integrin-induced signaling. Thromb Haemost82:385-91, 1999).

Calpains have been implicated in cell signaling through activation ofprotein kinases and phosphatases (cleaving between regulatory andcatalytic domains resulting in changes in activity after hydrolysis) andmodulation of their intracellular localization. Calpains have been shownto modify specific enzymes and cytoskeletal proteins as part ofcalcium-mediated signal pathways. They are also involved in remodelingand disassembling the cytoskeleton, especially where the cytoskeletonattaches to membranes or other subcellular structures.

Several nuclear transcription factors have been suggested as calpainsubstrates. Calpains are also involved in the progression of cellsthrough the cell cycle (Carafoli E and Molinari M Calpain: a protease insearch of a function? Biochem Biophys Res Commun 247:193-203, 1998) inthat calpain activity accelerates some cells through the cell cycle bycleavage of p53. Calpain is also thought to play a role in long termpotentiation (memory) and rat strains deficient in the endogenouscalpain inhibitor, calpastatin, have increased long term potentiation.

Calpains in Disease:

Several diseases have been associated with calpain deficiencies. Forexample, limb-girdle muscular dystrophy (LGMD) is a group of disordersthat primarily cause weakness of the shoulder and pelvic regions. Asubtype of LGMD called LGMD2A is caused by defects in the gene for p94(also called calpain 3) (Richard et al., Mutations in the proteolyticenzyme calpain 3 cause limb-girdle muscular dystrophy type 2A. Cell81:2740, 1995).

Positional cloning has recently identified single-nucleotidepolymorphisms (SNPs) in an intron of the gene coding for calpain-10 thatappears to confer insulin resistance in diabetics. Presence of thismutation correlates with reduced levels of calpain 10 in patientssusceptible to type II diabetes (Horikawa et al., Genetic variation inthe gene encoding calpain-10 is associated with type 2 diabetesmellitus. Nat Genet. 26:163-75, 2000). The same calpain-10 SNP alsocorrelates with type II diabetes in a high-risk population of PimaIndians (Baier et al., A calpain-10 gene polymorphism is associated withreduced muscle mRNA levels and insulin resistance. J Clin Invest.106:R69-73, 2000).

Over Activation of Calpain—Ischemic and Traumatic Damage

Intracellular calcium levels and calpain activity are normally tightlyregulated. Under stress, such as follows neuronal excitotoxicity,ischemic stroke, hemoragic stroke, hypoxic stress and/or trauma,intracellular calcium levels rise causing inappropriate calpainproteolytic activity. Calpain activity has been implicated in furthercell destruction and non-specific calpain inhibitors have been shown tobe protective in animal models (Lee et al., Proc. Natl. Acad. Sci. USA,88:7233-7237, 1991; Wang K K and Yuen P W. Calpain inhibition: anoverview of its therapeutic potential. Trends Pharmacol. Sci. 15:412-9,1994;

Lee, K S, et al., Calcium-activated proteolysis as a therapeutic targetin cerebrovascular disease. Annal NY Acad Sci. 825, 95-103, 1997).

Calpains are activated in neurons following ischemia-induced damage inanimal models of stroke. (Lee et al., Proc. Natl. Acad. Sci. USA,88:7233-7237, 1991). Inhibition of calcium-activated proteolysis bymeans of high doses of (usually non-specific) calpain inhibitors protectagainst the degeneration of vulnerable hippocampal neurons afterischemia (Rami et al., Brain Research, 609:67-70, 1993; Wang et al., Analpha-mercaptoacrylic acid derivative is a selective nonpeptidecell-permeable calpain inhibitor and is neuroprotective. Proc Natl AcadSci USA. 93:6687-92, 1996). After an ischemic insult, neuronal death isdelayed for hours to days. This time interval represents a potentialtherapeutic window in which to apply effective therapies to minimizebrain damage after stroke.

In addition to neuronal damage, calpains are thought to contribute tocardiac ischemic damage (Iwamoto H et al., Calpain inhibitor-1 reducesinfarct size and DNA fragmentation of myocardium in ischemic/reperfusedrat heart. J Cardiovasc Pharmacol 33:580-6, 1999) and hepatocytenecrosis during and following anoxia (Arora A S et al., Hepatocellularcarcinoma cells resist necrosis during anoxia by preventingphospholipase-mediated calpain activation. J Cell Physiol 167:434-42,1996).

Neurodegenerative Diseases

Calpains have been implicated in neurodegenerative diseases including,Alzheimer's disease, Multiple sclerosis, Huntington's disease,Parkinson's disease and amyotrophy. Calpain activation is increasedduring normal aging and a strong case can be made for the involvement ofcalpain in the abnormal proteolysis underlying the accumulation ofplaque and neurofibriles in brain tissue from people who sufferedAlzheimer-type dementia (Iwamoto et al., Brain Research, 561:177-1801991; Nixon et al., Calcium-activated neutral proteinase (calpain)system in aging and Alzheimer's disease. Ann N Y Acad Sci; 747:77-91,1994; Grynspan et al., Active site-directed antibodies identify calpainII as an early-appearing and pervasive component of neurofibrillarypathology in Alzheimer's disease. Brain Res 763:145-58, 1997). Calpainsare significantly activated in human postmortem brain from patients withAlzheimer's disease, and the degree of activation correlated with thoseregions of the brain showing the greatest amount of degeneration (Saitoet al., Proc. Natl. Acad. Sci. USA, 90:2628-2632, 1993). More recently,it has been recognized that in Alzheimer's disease cyclin-dependentkinase 5 (cdk5) and its neuron-specific activator p35 are involved inneurite outgrowth and cortical lamination. Calpain cleavage of p35produces p25, which accumulates in the brains of patients withAlzheimers disease. Conversion of p35 to p25 causes prolonged activationand mislocalization of cdk5 which hyperphosphorylates tau, disrupts thecytoskeleton and promotes the death (apoptosis) of primary neurons (Leeet al., Neurotoxicity induces cleavage of p35 to p25 by calpain. Nature.18;405:360-4, 2000). Compounds that inhibit calpain activity could proveuseful in reducing or delaying neurodegeneration caused to Alzheimer'sdisease.

Damage Following Trauma

Traumatic injury also causes calpain activation associated with furthercell death, atrophy and shrinkage of the brain. A forceful blow triggercell damage and increased calpain activity that can cleave structuralproteins in the brain for up to weeks afterward (Hayes et al., PotentialContribution of Proteases to Neuronal Damage Drug News & Perspectives11, 1998).

Calpain activation has also been implicated in spinal cord injuryfollowing trauma (for reviews see: Banik et al., Role of calpain and itsinhibitors in tissue degeneration and neuroprotection in spinal cordinjury. Ann. N. Y. Acad. Sci. 825:120-7 1997; Banik et al., Role ofcalpain in spinal cord injury: effects of calpain and free radicalinhibitors. Ann. N. Y. Acad. Sci. 844:131-7, 1998). Analogous to braintrauma, secondary pathophysiological alterations occur in thetraumatized spinal cord well after the initiating insult. Thesesecondary events ultimately cause cell death and tissue damage.Non-specific calpain inhibitors have shown utility in preventing furtherdamage due to spinal chord injury in animal models (Ray et al.,Increased calpain expression is associated with apoptosis in rat spinalcord injury: calpain inhibitor provides neuroprotection. Neurochem Res.25:1191-8, 2000).

These studies indicate the potential utility of calpain inhibitors(especially those calpains located in the spinal cord) in treatingtraumatic injury resulting from automobile crashes, gunshot wounds, andsports accidents.

Degeneration of Cochlear Tissues Following Noise Exposure

Calpains are activated during acoustic trauma and calpain inhibitorsprotect against hearing loss caused by noise (Stracher A Calpaininhibitors as therapeutic agents in nerve and muscle degeneration. Ann NY Acad Sci 884:52-9, 1999).

Inflammation

Calpains also regulate integrin-mediated interaction of T-cells with theextracellular matrix (ECM) and calpain inhibitors prevent acute andchronic inflammation in animal models (Cuzzocrea S et al., Calpaininhibitor I reduces the development of acute and chronic inflammation AmJ Pathol 157:2065-79, 2000).

Multiple Sclerosis

Multiple sclerosis is characterized by the progressive loss of themyelin of the brain and spinal cord. In autoimmune demyelinatingdiseases such as multiple sclerosis and experimental allergicencephalomyelitis, the degradation of myelin proteins results in thedestabilization of the myelin sheath. Calpains have been implicated inthat calpain degrades all major myelin proteins and increased calpainactivity is observed in multiple sclerosis (Shields D C et al., Aputative mechanism of demyelination in multiple sclerosis by aproteolytic enzyme, calpain. Proc. Natl. Acad. Sci. USA 96:11486-91,1999).

Cataract Formation

In the lens, crystallins prevent thermal denaturation and aggregation ofother proteins. Crystallins are also substrates for calpains andcataract formation in a rat model of selenite-induced cataract formationis thought to result from calpain activation and cleavage of crystallins(Shearer T R, David L L, Anderson R S, Azuma M. Review of selenitecataract. Curr Eye Res 1992; 11:357-369). In this model the crystallincleavage could be blocked by calpain inhibitors (Azuma M et al.,Cysteine protease inhibitor E64 reduces the rate of formation ofselenite cataract in the whole animal. Curr Eye Res 10:657-666, 1991).In a genetic model cataract-prone rats also showed enhanced proteolysisof crystallins and lens cytoskeletin proteins thought to be mediated bycalpain (Inomata M et al., Evidence for the involvement of calpain incataractogenesis,in Shumiya cataract rat (SCR). Biochim Biophys Acta1362:11-23 1997). Calpain activation is also thought to play a role incataracts induced by buthionine sulfoximine, calcium ionophore A23187,hydrogen peroxide, diamide, xylose, galactose and streptozotocin (Kadoyaet al., Role of calpain in hydrogen peroxide cataract. Curr Eye Res1993; 12:341-346; David et al., Buthionine sulfoximine induced cataractsin mice contain insolubilized crystallins with calpain II cleavagesites, Exp Eye Res 1994; 59:501-504.). These models of cataractformation in rats suggest that calpain-induced proteolysis is a commonunderlying mechanism. Fragments of alpha-crystallin, consistent withcalpain cleavage, have been also observed in cataractous human lens.

Reovirus Induced Myocarditis

Infection of neonatal mice with reovirus produces histologicalmyocarditis. This is due to a direct viral injury and apoptosis ofmyocytes. Calpain inhibitors block reovirus-induced apoptosis in vitroand prevented viral-induced induced myocarditis (DeBiasi et al., Calpaininhibition protects against virus-induced apoptotic myocardial injury.Virol 75:351-61, 2001).

The inventors of the present invention describe herein, thepolynucleotides corresponding to the full-length novel CAN-12 calpaingene, its encoded polypeptide, in addition to the variants CAN-12v1 andCAN-12v2. Also provided are polypeptide alignments illustrating thestrong conservation of the CAN-12, CAN-12v1, and CAN-12v2 polypeptidesto known proteases and a model of the active conformation of CAN-12.Based on this strong conservation, the inventors have ascribed theCAN-12, CAN-12v1, and CAN-12v2 polypeptides as having calpainproteolytic activity. Data is also provided illustrating the uniquetissue expression profile of the CAN-12 polypeptide in esophagus, lymphnode, and spinal cord tissues.

In fact, calpains have been the subject of significant research anddevelopment programs designed to identify inhibitors of this diseaseassociated protein class. For example, the following, non-limitingexamples of drugs, therapies, or regimens directed to inhibitingcalpains are currently known: BDA 410 (Mitsubishi Tokyo); AK 295(Alkermes; CAS® Registry Number: 160399-35-9, 144231-82-3, and145731-49-3;(1-(((1-ethyl-3-((3-(4-morpholinyl)propyl)amino)-2,3-dioxopropyl)amino)carbonyl)-3-methylbutyl)carbamicacid phenylmethyl ester stereois); AK 275 (Alkermes; CAS® RegistryNumber: 158798-83-5, and 150519-08-7;N-((phenylmethoxy)carbonyl)-L-leucyl-N-ethyl-L-2-aminobutanamide);inhibitor 1 (University of Indiana; acetyl-leu-leu-norleucinal);calpeptin (University of Indiana; benzyloxycarbonyl-leu-norleucinal);VASOLEX (Cortex); RESTENEX (Cortex); MDL 28170 (Aventis; CBZ-Val-Phe-H);P1 (Sankyo; CASS Registry Number: 128102-74-9, and 128102-75-0;L-phenylalanyl-L-glutaminyl-L-valyl-L-valyl-3-((3-nitro-2-pyridiny1)dithio)-L-alanylglycinamide); MDL 28170 (Hoechst Marion Roussel);BDA-410 (Mitsubishi-Tokyo); SJA-6017 (Senju; CAS® Registry Number:190274-534;Butanamide,2-(((4-fluorophenyl)sulfonyl)amino)-N-((1S)-1-formyl-3-methylbutyl.).-3-methyl-,(2S)-); Pharmaprojects No. 5123 (Pfizer; 2-Chloro-aceticacid(3-oxo-4-phenyl-3,4-dihydro-1H-quinoxalin-2-ylidene)hydrazide;WO96-25403); CEP-4143 (Cephalon; WO96-14067); MDL-104903 (Aventis; CAS)Registry Number: 180799-56-8; Carbamicacid,((1S)-1-(((4S,5R)-5-hydroxy-4-(phenylmethyl)-3-oxazolidinyl)carbonyl)-2-methylpropyl)-,phenylmethylester)); MDL-28170 (Aventis; CASS Registry Number: 19542-51-9; Alanine,N-(N-carboxy-L-valyl)-3-phenyl-N-benzyl ester, L-); CX-275 (CortexPharmaceuticals;PhenylmethylN-((1R)-1-(((1S)-1-ethyl-3-(ethylamino)-2,3-dioxopropyl)amino)carbonyl)-3-methylbutyl)carbamate); NS 7 (Nippon Shinyaku;4-(4-Fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy)pyrimidinehydrochloride ); Calpain inhibitor 1 (Suntory;N-Acetyl-L-leucinyl-L-leucinyl-L-norleucinal); E 64 (TaishoPharmaceutical (; CAS®) Registry Number: 66701-25-5); CEP 4143(Cephalon); SJA 6017 (Senju;N-(4-Fluorophenylsulfonyl)-L-valyl-L-leucinal); The present invention isdirected to antagonists specific to the CAN-12, CAN-12v1, and/orCAN-12v2 polypeptides. Modulating the activity of the calpainpolypeptides of the present invention may result in fewer toxicitiesthan the drugs, therapies, or regimens presently known to regulate otherknown calpains.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells, in addition to their use in theproduction of CAN-12, CAN-12v1, and CAN-12v2 polypeptides or peptidesusing recombinant techniques. Synthetic methods for producing thepolypeptides and polynucleotides of the present invention are provided.Also provided are diagnostic methods for detecting diseases, disorders,and/or conditions related to the CAN-12, CAN-12v1, and CAN-12v2polypeptides and polynucleotides, and therapeutic methods for treatingsuch diseases, disorders, and/or conditions. The invention furtherrelates to screening methods for identifying binding partners of thepolypeptides, particularly activators and inhibitors of the novelCAN-12, CAN-12v1, and CAN-12v2 polypeptides of the present invention.

BRIEF SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid molecules, thatcomprise, or alternatively consist of, a polynucleotide encoding theCAN-12 protein having the amino acid sequence shown in FIGS. 1A-E (SEQID NO:24) or the amino acid sequence encoded by the cDNA clone, CAN-12(also referred to as protease 5, clone 70).

The present invention provides isolated nucleic acid molecules, thatcomprise, or alternatively consist of, a polynucleotide encoding theCAN-12+ polypeptide sequence having the amino acid sequence shown inFIGS. 1A-E (SEQ ID NO:2) or the amino acid sequence encoded by the cDNAclone, CAN-12+ (also referred to as protease 5, clone 70; +splice aminoacids).

The present invention provides isolated nucleic acid molecules, thatcomprise, or alternatively consist of, a polynucleotide encoding theCAN-12v1 protein having the amino acid sequence shown in FIGS. 8A-C (SEQID NO:54) or the amino acid sequence encoded by the cDNA clone, CAN-12v1(also referred to as protease 5, clone 1e), deposited as ATCC DepositNumber PTA-3434 on Jun. 7, 2001.

The present invention provides isolated nucleic acid molecules, thatcomprise, or alternatively consist of, a polynucleotide encoding theCAN-12v2 protein having the amino acid sequence shown in FIGS. 9A-C (SEQID NO:56) or the amino acid sequence encoded by the cDNA clone, CAN-12v2(also referred to as protease 5, clone 1e1b-1), deposited as ATCCDeposit Number PTA-3434 on Jun. 7, 2001.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells, in addition to their use in theproduction of CAN-12, CAN-12v1, and CAN-12v1 polypeptides or peptidesusing recombinant techniques. Synthetic methods for producing thepolypeptides and polynucleotides of the present invention are provided.Also provided are diagnostic methods for detecting diseases, disorders,and/or conditions related to the CAN-12, CAN-12v1, and CAN-12v1polypeptides and polynucleotides, and therapeutic methods for treatingsuch diseases, disorders, and/or conditions. The invention furtherrelates to screening methods for identifying binding partners of thepolypeptides.

The invention further provides an isolated CAN-12 polypeptide having anamino acid sequence encoded by a polynucleotide described herein.

The invention further provides an isolated CAN-12v1 polypeptide havingan amino acid sequence encoded by a polynucleotide described herein.

The invention further provides an isolated CAN-12v2 polypeptide havingan amino acid sequence encoded by a polynucleotide described herein.

The invention also provides a machine readable storage medium whichcomprises the structure coordinates of CAN-12, including all or anyparts conserved calpain regions. Such storage medium encoded with thesedata are capable of displaying on a computer screen or similar viewingdevice, a three-dimensional graphical representation of a molecule ormolecular complex which comprises said regions or similarly shapedhomologous regions.

The invention also provides methods for designing, evaluating andidentifying compounds which bind to all or parts of the aforementionedregions. The methods include three dimensional model building (homologymodeling) and methods of computer assisted-drug design which can be usedto identify compounds which bind or modulate the forementioned regionsof the CAN-12 polypeptide. Such compounds are potential inhibitors ofCAN-12 or its homologues.

The invention also provides novel classes of compounds, andpharmaceutical compositions thereof, that are useful as inhibitors ofCAN-12 or its homologues.

The invention also provides novel classes of compounds, andpharmaceutical compositions thereof, that are useful as inhibitors ofCAN-12v1 or its homologues.

The invention also provides novel classes of compounds, andpharmaceutical compositions thereof, that are useful as inhibitors ofCAN-12v2 or its homologues.

The invention further relates to a polynucleotide encoding a polypeptidefragment of SEQ ID NO:2, 24, 54, and/or 56, or a polypeptide fragmentencoded by the cDNA sequence included in the deposited clone, which ishybridizable to SEQ ID NO:1, 23, 53, and/or 55.

The invention further relates to a polynucleotide encoding a polypeptidedomain of SEQ ID NO:2, 24, 54, and/or 56 or a polypeptide domain encodedby the cDNA sequence included in the deposited clone, which ishybridizable to SEQ ID NO:1, 23, 53, and/or 55.

The invention further relates to a polynucleotide encoding a polypeptideepitope of SEQ ID NO:2, 24, 54, and/or 56 or a polypeptide epitopeencoded by the cDNA sequence included in the deposited clone, which ishybridizable to SEQ ID NO:1, 23, 53, and/or 55.

The invention further relates to a polynucleotide encoding a polypeptideof SEQ ID NO:2, 24, 54, and/or 56 or the cDNA sequence included in thedeposited clone, which is hybridizable to SEQ ID NO:1, 23, 53, and/or55, having biological activity.

The invention further relates to a polynucleotide which is a variant ofSEQ ID NO:1, 23, 53, and/or 55.

The invention further relates to a polynucleotide which is an allelicvariant of SEQ ID NO:1, 23, 53, and/or 55.

The invention further relates to a polynucleotide which encodes aspecies homologue of the SEQ ID NO:2, 24, 54, and/or 56.

The invention further relates to a polynucleotide which represents thecomplimentary sequence (antisense) of SEQ ID NO:1, 23, 53, and/or 55.

The invention further relates to a polynucleotide capable of hybridizingunder stringent conditions to any one of the polynucleotides specifiedherein, wherein said polynucleotide does not hybridize under stringentconditions to a nucleic acid molecule having a nucleotide sequence ofonly A residues or of only T residues.

The invention further relates to an isolated nucleic acid molecule ofSEQ ID NO:2, 24, 54, and/or 56, wherein the polynucleotide fragmentcomprises a nucleotide sequence encoding a calpain protein.

The invention further relates to an isolated nucleic acid molecule ofSEQ ID NO:1, 23, 53, and/or 55 wherein the polynucleotide fragmentcomprises a nucleotide sequence encoding the sequence identified as SEQID NO:2, 24, 54, and/or 56 or the polypeptide encoded by the cDNAsequence included in the deposited clone, which is hybridizable to SEQID NO:1, 23, 53, and/or 55.

The invention further relates to an isolated nucleic acid molecule of ofSEQ ID NO:1, 23, 53, and/or 55 wherein the polynucleotide fragmentcomprises the entire nucleotide sequence of SEQ ID NO:1, 23, 53, and/or55 or the cDNA sequence included in the deposited clone, which ishybridizable to SEQ ID NO:1, 23, 53, and/or 55.

The invention further relates to an isolated nucleic acid molecule ofSEQ ID NO:1, 23, 53, and/or 55, wherein the nucleotide sequencecomprises sequential nucleotide deletions from either the C-terminus orthe N-terminus.

The invention further relates to an isolated polypeptide comprising anamino acid sequence that comprises a polypeptide fragment of SEQ IDNO:2, 24, 54, and/or 56 or the encoded sequence included in thedeposited clone.

The invention further relates to a polypeptide fragment of SEQ ID NO:2,24, 54, and/or 56 or the encoded sequence included in the depositedclone, having biological activity.

The invention further relates to a polypeptide domain of SEQ ID NO:2,24, 54, and/or 56 or the encoded sequence included in the depositedclone.

The invention further relates to a polypeptide epitope of SEQ ID NO:2,24, 54, and/or 56 or the encoded sequence included in the depositedclone.

The invention further relates to a full length protein of SEQ ID NO:2,24, 54, and/or 56 or the encoded sequence included in the depositedclone.

The invention further relates to a variant of SEQ ID NO:2, 24, 54,and/or 56.

The invention further relates to an allelic variant of SEQ ID NO:2, 24,54, and/or 56.

The invention further relates to a species homologue of SEQ ID NO:2, 24,54, and/or 56.

The invention further relates to the isolated polypeptide of of SEQ IDNO:2, 24, 54, and/or 56, wherein the full length protein comprisessequential amino acid deletions from either the C-terminus or theN-terminus.

The invention further relates to an isolated antibody that bindsspecifically to the isolated polypeptide of SEQ ID NO:2, 24, 54, and/or56.

The invention further relates to a method for preventing, treating, orameliorating a medical condition, comprising administering to amammalian subject a therapeutically effective amount of the polypeptideof SEQ ID NO:2, 24, 54, and/or 56 or the polynucleotide of SEQ ID NO:1,23, 53, and/or 55.

The invention further relates to a method of diagnosing a pathologicalcondition or a susceptibility to a pathological condition in a subjectcomprising the steps of (a) determining the presence or absence of amutation in the polynucleotide of SEQ ID NO:1, 23, 53, and/or 55; and(b) diagnosing a pathological condition or a susceptibility to apathological condition based on the presence or absence of saidmutation.

The invention further relates to a method of diagnosing a pathologicalcondition or a susceptibility to a pathological condition in a subjectcomprising the steps of (a) determining the presence or amount ofexpression of the polypeptide of of SEQ ID NO:2, 24, 54, and/or 56 in abiological sample; and diagnosing a pathological condition or asusceptibility to a pathological condition based on the presence oramount of expression of the polypeptide.

The invention further relates to a method for identifying a bindingpartner to the polypeptide of SEQ ID NO:2, 24, 54, and/or 56 comprisingthe steps of (a) contacting the polypeptide of SEQ ID NO:2, 24, 54,and/or 56 with a binding partner; and (b) determining whether thebinding partner effects an activity of the polypeptide.

The invention further relates to a gene corresponding to the cDNAsequence of SEQ ID NO:1, 23, 53, and/or 55.

The invention further relates to a method of identifying an activity ina biological assay, wherein the method comprises the steps of expressingSEQ ID NO:1, 23, 53, and/or 55 in a cell, (b) isolating the supernatant;(c) detecting an activity in a biological assay; and (d) identifying theprotein in the supernatant having the activity.

The invention further relates to a process for making polynucleotidesequences encoding gene products having altered activity selected fromthe group consisting of SEQ ID NO:2, 24, 54, and/or 56 activitycomprising the steps of (a) shuffling a nucleotide sequence of SEQ IDNO:1, 23, 53, and/or 55, (b) expressing the resulting shufflednucleotide sequences and, (c) selecting for altered activity selectedfrom the group consisting of SEQ ID NO:2, 24, 54, and/or 56 activity ascompared to the activity selected from the group consisting of SEQ IDNO:2, 24, 54, and/or 56 activity of the gene product of said unmodifiednucleotide sequence.

The invention further relates to a shuffled polynucleotide sequenceproduced by a shuffling process, wherein said shuffled DNA moleculeencodes a gene product having enhanced tolerance to an inhibitor of anyone of the activities selected from the group consisting of SEQ ID NO:2,24, 54, and/or 56 activity.

The invention further relates to a method for preventing, treating, orameliorating a medical condition with the polypeptide provided as SEQ IDNO:2, 24, 54, and/or 56, in addition to, its encoding nucleic acid,wherein the medical condition is a gastrointenstinal disorder.

The invention further relates to a method for preventing, treating, orameliorating a medical condition with the polypeptide provided as SEQ IDNO:2, 24, 54, and/or 56, in addition to, its encoding nucleic acid,wherein the medical condition is a neural disorder.

The invention further relates to a method for preventing, treating, orameliorating a medical condition with the polypeptide provided as SEQ IDNO:2, 24, 54, and/or 56, in addition to, its encoding nucleic acid,wherein the medical condition is an inflammatory disease.

The invention further relates to a method for preventing, treating, orameliorating a medical condition with the polypeptide provided as SEQ IDNO:2, 24, 54, and/or 56, in addition to, its encoding nucleic acid,wherein the medical condition is an inflammatory disease whereproteases, either directly or indirectly, are involved in diseaseprogression.

The invention further relates to a method for preventing, treating, orameliorating a medical condition with the polypeptide provided as SEQ IDNO:2, 24, 54, and/or 56, in addition to, its encoding nucleic acid,wherein the medical condition is a degenerative disease whereinproteases, either directly or indirectly, are involved in diseaseprogression.

The invention further relates to a method for preventing, treating, orameliorating a medical condition with the polypeptide provided as SEQ IDNO:2, 24, 54, and/or 56, in addition to, its encoding nucleic acid,wherein the medical condition is multiple sclerosis.

The invention further relates to a method for preventing, treating, orameliorating a medical condition with the polypeptide provided as SEQ IDNO:2, 24, 54, and/or 56, in addition to, its encoding nucleic acid,wherein the medical condition is a cancer.

The invention further relates to a method for preventing, treating, orameliorating a medical condition with the polypeptide provided as SEQ IDNO:2, 24, 54, and/or 56, in addition to, its encoding nucleic acid,wherein the medical condition is a blood disorder.

The invention further relates to a method for preventing, treating, orameliorating a medical condition with the polypeptide provided as SEQ IDNO:2, 24, 54, and/or 56, in addition to, its encoding nucleic acid,wherein the medical condition is an immune disorder.

The invention further relates to a method for preventing, treating, orameliorating a medical condition with the polypeptide provided as SEQ IDNO:2, 24, 54, and/or 56, in addition to, its encoding nucleic acid,wherein the medical condition is a hematopoietic disorder.

The invention further relates to a method for preventing, treating, orameliorating a medical condition with the polypeptide provided as SEQ IDNO:2, 24, 54, and/or 56, in addition to, its encoding nucleic acid,wherein the medical condition is a disorder related to aberrant proteaseregulation.

The invention further relates to a method for preventing, treating, orameliorating a medical condition with the polypeptide provided as SEQ IDNO:2, 24, 54, and/or 56, in addition to, its encoding nucleic acid,wherein the medical condition is a condition associated with tissuedamage caused by calpain activation, either directly or indirectly.

The invention further relates to a method of identifying a compound thatmodulates the biological activity of CAN-12, comprising the steps of,(a) combining a candidate modulator compound with CAN-12 having thesequence set forth in one or more of SEQ ID NO:2, 24, 54, and/or 56; andmeasuring an effect of the candidate modulator compound on the activityof CAN-12.

The invention further relates to a method of identifying a compound thatmodulates the biological activity of a calpain, comprising the steps of,(a) combining a candidate modulator compound with a host cell expressingCAN-12 having the sequence as set forth in SEQ ID NO:2, 24, 54, and/or56; and, (b) measuring an effect of the candidate modulator compound onthe activity of the expressed CAN-12.

The invention further relates to a method of identifying a compound thatmodulates the biological activity of CAN-12, comprising the steps of,(a) combining a candidate modulator compound with a host cell containinga vector described herein, wherein CAN-12 is expressed by the cell; and,(b) measuring an effect of the candidate modulator compound on theactivity of the expressed CAN-12.

The invention further relates to a method of screening for a compoundthat is capable of modulating the biological activity of CAN-12,comprising the steps of: (a) providing a host cell described herein; (b)determining the biological activity of CAN-12 in the absence of amodulator compound; (c) contacting the cell with the modulator compound;and (d) determining the biological activity of CAN-12 in the presence ofthe modulator compound; wherein a difference between the activity ofCAN-12 in the presence of the modulator compound and in the absence ofthe modulator compound indicates a modulating effect of the compound.

The invention further relates to a compound that modulates thebiological activity of human CAN-12 as identified by the methodsdescribed herein.

The invention also provides a machine readable storage medium whichcomprises the structure coordinates of CAN-12, including all or anyparts conserved calpain regions. Such storage medium encoded with thesedata are capable of displaying on a computer screen or similar viewingdevice, a three-dimensional graphical representation of a molecule ormolecular complex which comprises said regions or similarly shapedhomologous regions.

The invention also provides a machine-readable data storage medium,comprising a data storage material encoded with machine readable data,wherein the data is defined by the structure coordinates of the modelCAN-12 according to Table IV or a homologue of said model, wherein saidhomologue comprises any kind of surrogate atoms that have a root meansquare deviation from the backbone atoms of the complex of not more thanabout 5.0 Å.

The invention also provides a machine-readable data storage medium,comprising a data storage material encoded with machine readable data,wherein the data is defined by the structure coordinates of the modelCAN-12 according to Table IV or a homologue of said model, wherein saidhomologue comprises any kind of surrogate atoms that have a root meansquare deviation from the backbone atoms of the complex of not more thanabout 5.0 Å, preferably not more than about 4.0 Å, or less.

The invention also provides a model comprising all or any part of themodel defined by structure coordinates of CAN-12 according to Table IV,or a mutant or homologue of said molecule or molecular complex.

The invention also provides a method for identifying a mutant of CAN-12with altered biological properties, function, or reactivity, the methodcomprising one or more of the following steps: (a) use of the model or ahomologue of said model according to Table IV, for the design of proteinmutants with altered biological function or properties which exhibit anycombination of therapeutic effects described herein; and/or (b) use ofthe model or a homologue of said model, for the design of a protein withmutations in the active site region comprised of the amino acids fromabout amino acid R90 to about amino acid C94, from about amino acid G99to about amino acid L104, from about amino acid Q196 to about amino acidS198, from about amino acid M210 to about amino acid I212, from aboutamino acid G236 to about amino acid H240, from about amino acid E251 toabout amino acid Y255, from about amino acid N277 to about amino acidK281, from about amino acid V327 to about amino acid V330 of SEQ ID NO:2according to Table IV with altered biological function or propertieswhich exhibit any combination of therapeutic effects described herein.

The invention also provides methods for designing, evaluating andidentifying compounds which bind to all or parts of the aforementionedregions. The methods include three dimensional model building (homologymodeling) and methods of computer assisted-drug design which can be usedto identify compounds which bind or modulate the forementioned regionsof the CAN-12 polypeptide. Such compounds are potential inhibitors ofCAN-12 or its homologues.

The invention also relates to method for identifying modulators ofCAN-12 biological properties, function, or reactivity, the methodcomprising the step of modeling test compounds that fit spatially intothe EF-hand calcium binding region defined by I449-K471 of SEQ ID NO:2using a homologue or portion thereof or analogue in which the originalC, N, and O atoms have been replaced with other elements

The invention also relates to a method of using said structurecoordinates as set forth in Table IV to identify structural and chemicalfeatures of CAN-12; employing identified structural or chemical featuresto design or select compounds as potential CAN-12 modulators; employingthe three-dimensional structural model to design or select compounds aspotential CAN-12 modulators; synthesizing the potential CAN-12modulators; screening the potential CAN-12 modulators in an assaycharacterized by binding of a protein to the CAN-12. The invention alsorelates to said method wherein the potential CAN-12 modulator isselected from a database. The invention further relates to said methodwherein the potential CAN-12 modulator is designed de novo. Theinvention further relates to a method wherein the potential CAN-12modulator is designed from a known modulator of activity.

The invention also provides a machine-readable data storage medium,comprising a data storage material encoded with machine readable data,wherein the data is defined by the structure coordinates of the modelCAN-12v2v2 according to Table V or a homologue of said model, whereinsaid homologue comprises any kind of surrogate atoms that have a rootmean square deviation from the backbone atoms of the complex of not morethan 5.0 Å.

The invention also provides a machine-readable data storage medium,comprising a data storage material encoded with machine readable data,wherein the data is defined by the structure coordinates of the modelCAN-12v2 according to Table V or a homologue of said model, wherein saidhomologue comprises any kind of surrogate atoms that have a root meansquare deviation from the backbone atoms of the complex of not more than5.0 Å, preferabbly not more than 4.0 Å, or less

The invention also provides a model comprising all or any part of themodel defined by structure coordinates of CAN-12v2 according to Table V,or a mutant or homologue of said molecule or molecular complex.

The invention also provides a method for identifying a mutant ofCAN-12v2 with altered biological properties, function, or reactivity,the method comprising one or more of the following steps: (a) use of themodel or a homologue of said model according to Table V, for the designof protein mutants with altered biological function or properties whichexhibit any combination of therapeutic effects described herein; and/or(b) use of the model or a homologue of said model, for the design of aprotein with mutations in the active site region comprised of the aminoacids from about amino acid R90 to about amino acid C94, from aboutamino acid G99 to about amino acid L104, from about amino acid Q196 toabout amino acid S198, from about amino acid M210 to about amino acidI212, from about amino acid G236 to about amino acid H240, from aboutamino acid E252 to about amino acid Y256, from about amino acid N278 toabout amino acid K282, from about amino acid V328 to about amino acidV331 of SEQ ID NO:56 according to Table V with altered biologicalfunction or properties which exhibit any combination of therapeuticeffects described herein.

The invention also provides methods for designing, evaluating andidentifying compounds which bind to all or parts of the aforementionedregions. The methods include three dimensional model building (homologymodeling) and methods of computer assisted-drug design which can be usedto identify compounds which bind or modulate the forementioned regionsof the CAN-12v2 polypeptide. Such compounds are potential inhibitors ofCAN-12v2 or its homologues.

The invention also relates to method for identifying modulators ofCAN-12v2 biological properties, function, or reactivity, the methodcomprising the step of modeling test compounds that fit spatially intothe EF-hand calcium binding regions defined by amino acids 1565 to K587of SEQ ID NO:56, using a homologue or portion thereof or analogue inwhich the original C, N, and O atoms have been replaced with otherelements The invention also relates to a method of using said structurecoordinates as set forth in Table V to identify structural and chemicalfeatures of CAN-12v2; employing identified structural or chemicalfeatures to design or select compounds as potential CAN-12v2 modulators;employing the three-dimensional structural model to design or selectcompounds as potential CAN-12v2 modulators; synthesizing the potentialCAN-12v2 modulators; screening the potential CAN-12v2 modulators in anassay characterized by binding of a protein to the CAN-12v2. Theinvention also relates to said method wherein the potential CAN-12v2modulator is selected from a database. The invention further relates tosaid method wherein the potential CAN-12v2 modulator is designed denovo. The invention further relates to a method wherein the potentialCAN-12v2 modulator is designed from a known modulator of activity.

The present invention also relates to an isolated polynucleotideconsisting of a portion of the human CAN-12 gene consisting of at least8 bases, specifically excluding Genbank Accession Nos. gi|AL540944,and/or gi|BM554389.

The present invention also relates to an isolated polynucleotideconsisting of a nucleotide sequence encoding a fragment of the humanCAN-12 protein, wherein said fragment displays one or more functionalactivities specifically excluding Genbank Accession Nos. gi|AL540944,and/or gi|BM554389.

The present invention also relates to the polynucleotide of SEQ ID NO:1,23, 53, and/or 55 consisting of at least 10 to 50 bases, wherein said atleast 10 to 50 bases specifically exclude the polynucleotide sequence ofGenbank Accession Nos. gi|AL540944, and/or gi|BM554389.

The present invention also relates to the polynucleotide of SEQ ID NO:1,23, 53, and/or 55 consisting of at least 15 to 100 bases, wherein saidat least 15 to 100 bases specifically exclude the polynucleotidesequence of Genbank Accession Nos. gi|AL540944, and/or gi|BM554389.

The present invention also relates to the polynucleotide of SEQ ID NO:1,23, 53, and/or 55 consisting of at least 100 to 1000 bases, wherein saidat least 100 to 1000 bases specifically exclude the polynucleotidesequence of Genbank Accession Nos. gi|AL540944, and/or gi|BM554389.

The present invention also relates to an isolated polypeptide fragmentof the human CAN-12 protein, wherein said polypeptide fragment does notconsist of the polypeptide encoded by the polynucleotide sequence ofGenbank Accession Nos. gi|AL540944, and/or gi|BM554389.

BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS

The file of this patent contains at least one Figure executed in color.Copies of this patent with color Figure(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIGS. 1A-E show the polynucleotide sequence (SEQ ID NO:1) and deducedamino acid sequence (SEQ ID NO:24) of the novel human calpain, CAN-12,of the present invention. The standard one-letter abbreviation for aminoacids is used to illustrate the deduced amino acid sequence. Thepolynucleotide sequence contains a sequence of 4584 nucleotides (SEQ IDNO:1), encoding a polypeptide of about 428 amino acids (SEQ ID NO:24).The polynucleotide sequence of CAN-12 is believed to represent a shortsplice variant form. As a result, the alternative splicing introduces astop codon truncating the open reading frame to end at amino acid 428.Additional amino acids beyond amino acid 428 of SEQ ID NO:24 are shownand are represented in bold (beginning at nucleotide 1537 to 1995 of SEQID NO:1). These additional amino acids likely corresponded to thepolypeptide sequence of alternative splice forms of CAN-12 as evidencedby the presence of the EF-hand calcium binding domain. However, theseadditional amino acids are not considered to be a part of this spliceform (SEQ ID NO:24). Additional splice forms of CAN-12 have beenidentified and are described herein (CAN-12v1 and CAN-12v2). The CAN-12polypeptide sequence comprising these additional amino acids is providedas SEQ ID NO:2 to serve as a reference for the CAN-12v1 and CAN12-v2splice variants. An analysis of the CAN-12 polypeptide determined thatit comprised the following features: predicted active site domain aminoacids located from about amino acid R90 to about amino acid C94, fromabout amino acid G99 to about amino acid L104, from about amino acidQ196 to about amino acid S198, from about amino acid M2 10 to aboutamino acid I212, from about amino acid G236 to about amino acid H240,from about amino acid E251 to about amino acid Y255, from about aminoacid N277 to about amino acid K281, and/or from about amino acid V327 toabout amino acid V330 of SEQ ID NO:24 (FIGS. 1A-E) represented byshading; and a predicted eukaryotic thiol (cysteine) protease activesite domain located from about amino acid 90 to about amino acid 111 ofSEQ ID NO:24 (FIGS. 1A-E) represented by double underlining. Thepredicted active site domain amino acids are believed to form the activesite binding pocket of the CAN-12 polypeptide and facilitate catalysisof appropriate calpain substrates. The predicted catalytic amino acidresidues within the CAN-12 active site are located at amino acid C101,H253, and N277 residues of SEQ ID NO:24 (FIGS. 1A-E) and are denoted byan arrow (“T”). The additional amino acids beyond amino acid 428 of SEQID NO:24 depicted in the Figure were predicted to comprise an EF-handcalcium-binding domain located from about amino acid 439 to about aminoacid 471 of SEQ ID NO:24 (FIGS. 1A-E) represented by dotted underlining;and a predicted cell attachment sequence located from about amino acid520 to about amino acid 532 of SEQ ID NO:24 (FIGS. 1A-E) represented initalics. The presence of the eukaryotic thiol (cysteine) protease activesite domain in the additional translated amino acids supports the notionthat additional splice variants of CAN-12 exist, at least two of whichare described herein.

FIGS. 2A-E show the regions of identity and similarity between theencoded CAN-12 (SEQ ID NO:2), CAN-12v1 (SEQ ID NO:54), and CAN-12v2polypeptides (SEQ ID NO:56) to other calpains, specifically, the humanCAN10 protein (type II diabetes linked) (hCAN10; Genbank Accession No:gi|NP_(—)075574; SEQ ID NO:3); the human CAN5 protein (hCAN5; GenbankAccession No: gi|NP_(—)004046; SEQ ID NO:4); the large catalytic subunitof the human CALPAIN 1 protein (also referred to as Calcium-ActivatedNeutral Proteinase, CANP, μ-TYPE) (hCAN1; Genbank Accession No:gi|12408656; SEQ ID NO:5); the large subunit of the human calpain 3protein (EC 3.4.22.17) (also referred to as CALPAIN L3, CALPAIN P94,Calcium-Activated Neutral Proteinase 3, CANP 3; muscle-specificcalcium-activated neutral protease 3 large subunit) (hCAN3; GenbankAccession No: gi|4557405; SEQ ID NO:6); the large catalytic subunit ofthe mouse CALPAIN 1 protein (also referred to as Calcium-ActivatedNeutral Proteinase) (CANP) G-TYPE ) (mCALPAIN1; Genbank Accession No:gi|O88666; SEQ ID NO:7); the mouse CALPAIN LP82 (mLP82; GenbankAccession No: gi|3661585; SEQ ID NO:8); the rat stomach-specificcalcium-activated neutral protease large subunit, NCL2 protein (rNCL2;Genbank Accession No: gi|NP_(—)006606; SEQ ID NO:9); the human CAN11protein (hCAN11; Genbank Accession No: gi|NP_(—)008989; SEQ ID NO:10);the human CAN2 protein (hCAN2; Genbank Accession No: gi|4502563; SEQ IDNO:11); and the human CAN9 protein (hCAN9; Genbank Accession No:gi|5729758; SEQ ID NO:12). The alignment was performed using theCLUSTALW algorithm described elsewhere herein. The darkly shaded aminoacids represent regions of matching identity. The lightly shaded aminoacids represent regions of matching similarity. Lines between residuesindicate gapped regions for the aligned polypeptides. The asterisk (“↑”)denotes the characteristic active site cysteine (C101), histidine(H253), and asparagine (N277) residues of calpain proteases. The CAN-12polypeptide sequence shown (CAN12+; SEQ ID NO:2) includes the additionaltranslated amino acids beyond amino acid 428 of SEQ ID NO:24 (shown inFIGS. 1A-E) to illustrate their identity with the CAN-12v1 and CAN-12v2splice variants.

FIG. 3 shows a phylogenetic tree organization of various calpain familymembers with respect to the CAN-12 polypeptide of the present invention.The organization was created using the Vector NTI AlignX algorithm,based upon the CLUSTALW alignment described in FIGS. 2A-E above. Asshown, CAN-12 is most closely related, phylogenetically, to the humanCAN5 and CAN10 proteins.

FIG. 4 shows an expression profile of the novel human calpain, CAN-12.The figure illustrates the relative expression level of CAN-12 amongstvarious mRNA tissue sources. As shown, transcripts corresponding toCAN-12 expressed highly in spinal cord. The CAN-12 polypeptide was alsoexpressed significantly in lymph node, thymus, and to a lesser extent,in spleen. Expression data was obtained by measuring the steady stateCAN-12 mRNA levels by quantitative PCR using the PCR primer pairprovided as SEQ ID NO:21 and 22 as described herein.

FIGS. 5A-B show a table illustrating the percent identity and percentsimilarity values between the CAN-12+ (SEQ ID NO:2), CAN-12v1 (SEQ IDNO:54), CAN-12v2 (SEQ ID NO:56), and CAN-12 (SEQ ID NO:24) polypeptidesof the present invention with other calpains, specifically, the humanCAN10 protein (type II diabetes linked) (hCAN10; Genbank Accession No:gi|NP_(—)075574; SEQ ID NO:3); the human CAN5 protein (hCAN5; GenbankAccession No: gi|NP_(—)004046; SEQ ID NO:4); the large catalytic subunitof the human CALPAIN 1 protein (also referred to as Calcium-ActivatedNeutral Proteinase, CANP, μ-TYPE) (hCAN1; Genbank Accession No:gi|12408656; SEQ ID NO:5); the large subunit of the human calpain 3protein (EC 3.4.22.17) (also referred to as CALPAIN L3, CALPAIN P94,Calcium-Activated Neutral Proteinase 3, CANP 3; muscle-specificcalcium-activated neutral protease 3 large subunit) (hCAN3; GenbankAccession No: gi|4557405; SEQ ID NO:6); the large catalytic subunit ofthe mouse CALPAIN 1 protein (also referred to as Calcium-ActivatedNeutral Proteinase) (CANP) μ-TYPE ) (mCALPAIN1; Genbank Accession No:gi|088666; SEQ ID NO:7); the mouse CALPAIN LP82 (mLP82; GenbankAccession No: gi|3661585; SEQ ID NO:8); the rat stomach-specificcalcium-activated neutral protease large subunit, NCL2 protein (rNCL2;Genbank Accession No: gi|NP_(—)006606; SEQ ID NO:9); the human CAN11protein (hCAN11; Genbank Accession No: gi|NP_(—)008989; SEQ ID NO:10);the human CAN2 protein (hCAN2; Genbank Accession No: gi|4502563; SEQ IDNO:11); and the human CAN9 protein (hCAN9; Genbank Accession No:gi|5729758; SEQ ID NO:12). The alignment was performed using theCLUSTALW algorithm described elsewhere herein. The darkly shaded aminoacids represent regions of matching identity. The lightly shaded aminoacids represent regions of matching similarity. Lines between residuesindicate gapped regions for the aligned polypeptides.

FIGS. 6 shows a three-dimensional homology model of the CAN-12polypeptide based upon the homologous structure of a portion of thehuman m-calpain, also referred to as, CAN2 (hCAN2; Genbank Accession No.gi|4502563; SEQ ID NO:11). The predicted catalytic active site aminoacids of the human CAN-12 polypeptide are labeled. The predicted regionsof alpha helix structure are represented in magenta; the predictedregions of beta sheet structure are represented in yellow; the predictedregions of flexible loop structure are represented in cyan; thecatalytic amino acid residues are shown in a CPK/space filled renderingof the side chain atoms wherein carbon atoms are represented in white,the sulfur atoms are represented in yellow, and the nitrogen atoms arerepresented in blue. The structural coordinates of the CAN-12polypeptide are provided in Table IV herein. The homology model ofCAN-12 was derived from generating a sequence alignment with the humanm-calpain, CAN2 protein (hCAN2; Genbank Accession No. gi|4502563; SEQ IDNO:11) using the Proceryon suite of software (Proceryon Biosciences,Inc. N.Y., N.Y.), and the overall atomic model including plausiblesidechain orientations using the program LOOK (V3.5.2, MolecularApplications Group).

FIGS. 8A-C show the polynucleotide sequence (SEQ ID NO:53) and deducedamino acid sequence (SEQ ID NO:54) of the novel human calpain, CAN-12v1,of the present invention. The standard one-letter abbreviation for aminoacids is used to illustrate the deduced amino acid sequence. Thepolynucleotide sequence contains a sequence of 2095 nucleotides (SEQ IDNO:53), encoding a polypeptide of about 694 amino acids (SEQ ID NO:54).The polynucleotide sequence of CAN-12v1 is believed to represent a novelsplice variant of the CAN-12 polynucleotide described herein. Ananalysis of the CAN-12v1 polypeptide determined that it comprised thefollowing features: predicted active site domain amino acids locatedfrom about amino acid R90 to about amino acid C94, from about amino acidG99 to about amino acid L104, from about amino acid Q196 to about aminoacid S198, from about amino acid M210 to about amino acid I212, fromabout amino acid G236 to about amino acid H240, from about amino acidE252 to about amino acid Y256, from about amino acid N278 to about aminoacid K282, and/or from about amino acid V328 to about amino acid V331 ofSEQ ID NO:54 (FIGS. 8A-C) represented by shading; a predicted eukaryoticthiol (cysteine) protease active site domain located from about aminoacid 90 to about amino acid 111 of SEQ ID NO:54 (FIGS. 8A-C) representedby double underlining; a predicted EF-hand calcium-binding domainlocated from about amino acid 567 to about amino acid 584 of SEQ IDNO:54 (FIGS. 8A-C) represented by dotted underlining; and a predictedcell attachment sequence located from about amino acid 633 to aboutamino acid 532 of SEQ ID NO:54 (FIGS. 8A-C) represented in italics. Thepresence of the eukaryotic thiol (cysteine) protease active site domain,in addition to, the EF-hand calcium binding domain is consistent withthe CAN-12v1 polypeptide representing a member of the calpain family ofproteases. The predicted active site domain amino acids are believed toform the active site binding pocket of the CAN-12v1 polypeptide andfacilitate catalysis of appropriate calpain substrates. The predictedcatalytic amino acid residues within the CAN-12v1 active site arelocated at amino acid C101, H254, and N278 residues of SEQ ID NO:54(FIGS. 8A-C) and are denoted by an arrow (“↑”).

FIGS. 9A-C show the polynucleotide sequence (SEQ ID NO:55) and deducedamino acid sequence (SEQ ID NO:56) of the novel human calpain, CAN-12v2,of the present invention. The standard one-letter abbreviation for aminoacids is used to illustrate the deduced amino acid sequence. Thepolynucleotide sequence contains a sequence of 2104 nucleotides (SEQ IDNO:53), encoding a polypeptide of about 694 amino acids (SEQ ID NO:56).The polynucleotide sequence of CAN-12v2 is believed to represent a novelsplice variant of the CAN-12 polynucleotide described herein and likelyrepresents the physiologically relevant splice form. An analysis of theCAN-12v2 polypeptide determined that it comprised the followingfeatures: predicted active site domain amino acids located from aboutamino acid R90 to about amino acid C94, from about amino acid G99 toabout amino acid L104, from about amino acid Q196 to about amino acidS198, from about amino acid M210 to about amino acid I212, from aboutamino acid G236 to about amino acid H240, from about amino acid E252 toabout amino acid Y256, from about amino acid N278 to about amino acidK282, and/or from about amino acid V328 to about amino acid V331 of SEQID NO:56 (FIGS. 9A-C) represented by shading; a predicted eukaryoticthiol (cysteine) protease active site domain located from about aminoacid 90 to about amino acid 111 of SEQ ID NO:56 (FIGS. 9A-C) representedby double underlining; a predicted EF-hand calcium-binding domainlocated from about amino acid 565 to about amino acid 587 of SEQ IDNO:56 (FIGS. 9A-C) represented by dotted underlining; and a predictedcell attachment sequence located from about amino acid 636 to aboutamino acid 648 of SEQ ID NO:56 (FIGS. 9A-C) represented by italics. Thepresence of the eukaryotic thiol (cysteine) protease active site domain,in addition to, the EF-hand calcium binding domain is consistent withthe CAN-12v2 polypeptide representing a member of the calpain family ofproteases. The predicted active site domain amino acids are believed toform the active site binding pocket of the CAN-12v2 polypeptide andfacilitate catalysis of appropriate calpain substrates. The predictedcatalytic amino acid residues within the CAN-12v2 active site arelocated at amino acid C101, H254, and N278 residues of SEQ ID NO:56(FIGS. 9A-C) and are denoted by an arrow (“↑”).CAN-12v2 is believed torepresent the true physioligical form of CAN-12.

FIGS. 10A-B show the regions of identity and similarity between theencoded CAN-12+ (SEQ ID NO:2), CAN-12v1 (SEQ ID NO:54), CAN-12v2polypeptides (SEQ ID NO:56), and CAN-12 (SEQ ID NO:24) The alignment wasperformed using the CLUSTALW algorithm described elsewhere herein. Thedarkly shaded amino acids represent regions of matching identity. Thelightly shaded amino acids represent regions of matching similarity.Lines between residues indicate gapped regions for the alignedpolypeptides.

FIGS. 11 shows a three-dimensional homology model of the CAN-12v2polypeptide based upon the homologous structure of a portion of thehuman m-calpain, also referred to as, CAN2 (hCAN2; Genbank Accession No.gi|4502563; SEQ ID NO:11). The predicted catalytic active site aminoacids of the human CAN-12v2 polypeptide are labeled. The predictedregions of alpha helix structure are represented in magenta; thepredicted regions of beta sheet structure are represented in yellow; thepredicted regions of flexible loop structure are represented in cyan;the catalytic amino acid residues are shown in a CPK/space filledrendering of the side chain atoms wherein carbon atoms are representedin white, the sulfur atoms are represented in yellow, and the nitrogenatoms are represented in blue. The structural coordinates of theCAN-12v2 polypeptide are provided in Table V herein. The homology modelof CAN-12v2 was derived from generating a sequence alignment with thehuman m-calpain, CAN2 protein (hCAN2; Genbank Accession No. gi|4502563;SEQ ID NO:11) using the SYBYL suite of software (Tripos, Inc., St.Louis, Mo.), and the overall atomic model including plausible sidechainorientations using the program COMPOSER (Tripos, Inc., St. Louis, Mo.).

FIG. 12 shows an expanded expression profile of the novel human calpain,CAN-12. The figure illustrates the relative expression level of CAN-12amongst various mRNA tissue sources. As shown, the CAN-12 polypeptidewas expressed at relatively low levels, though predominately ineosphagus, lymph node, and to a lesser extent in other tissues as shown.Expression data was obtained by measuring the steady state CAN-12 mRNAlevels by quantitative PCR using the PCR primer pair provided as SEQ IDNO:143 and 144, and Taqman probe (SEQ ID NO:145) as described in Example6 herein.

FIG. 13 shows an energy graph for the CAN-12.v2 model (see FIG. 11) ofthe present invention (solid line) and the human m-calpain template (PDBcode 1dkv) (dotted line) from which the model was generated. The energydistribution for each protein fold is displayed on the y-axis, while theamino acid residue position of the protein fold is displayed on thex-axis. As shown, the CAN-12.v2 model and 1dkv template have similarenergies over the aligned region, suggesting that the structural modelof CAN-12.v2 represents a “native-like” conformation of the CAN-12.v2polypeptide. This graph supports the motif and sequence alignments inconfirming that the three-dimensional structure coordinates of CAN-12.v2are an accurate and useful representation of the structure of theCAN-12.v2 polypeptide.

Table I provides a summary of the novel polypeptides and their encodingpolynucleotides of the present invention.

Table II illustrates the preferred hybridization conditions for thepolynucleotides of the present invention. Other hybridization conditionsmay be known in the art or are described elsewhere herein.

Table III provides a summary of various conservative substitutionsencompassed by the present invention.

Table IV provides the structural coordinates of the homology model ofthe CAN-12 polypeptide provided in FIG. 6. A description of the headingsare as follows: “Atom No” refers to the atom number within the CAN-12homology model; “Atom Name” refers to the element whose coordinates aremeasured, the first letter in the column defines the element; “Residue”refers to the amino acid of the CAN-12 polypeptide within which the atomresides, in addition to the amino acid position in which the atomresides; “X Coord”, “Y Coord”, and “Z Coord” structurally define theatomic position of the element measured in three dimensions.

Table V provides the structural coordinates of the homology model of theCAN-12v2 polypeptide provided in FIG. 11. A description of the headingsare as follows: “Atom No” refers to the atom number within the CAN-12v2homology model; “Atom Name” refers to the element whose coordinates aremeasured, the first letter in the column defines the element; “Residue”refers to the amino acid of the CAN-12v2 polypeptide within which theatom resides, in addition to the amino acid position in which the atomresides; “X Coord”, “Y Coord”, and “Z Coord” structurally define theatomic position of the element measured in three dimensions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the Examples included herein. All references to “CAN-12”shall be construed to apply to CAN-12+, CAN-12, CAN-12v1, and/orCAN-12v2 unless otherwise specified herein.

The invention provides a novel human sequence that encodes a calpainwith substantial homology to the large subunits of a variety of knowncalpains. Calpains affect a variety of cellular processes based upontheir involvement in modulating signal transduction. Aberrations in thelarge subunit polypeptides of calpains have been implicated in a numberof diseases and disorders which include, for example, incidence of typeII diabetes (Horikawa et al., Nat Genet. 26:163-75 (2000)), limb-girdlemuscular dystrophy (Richard et al., Cell 81:27-40 (1995)),ischemia-induced damage in neurons and heart tissue, neurodegnerativedisorders such as Alzheimer's disease, Multiple sclerosis, Huntington'sdisease, Parkinson's disease and amyotrophy, inflammatory disorders,susceptibility to infectious diseases, etc. CAN-12 polynucleotides andpolypeptides, including agonists and antagonists thereof are expected tobe useful in ameliorating at least some of these disorders. In addition,expression analysis indicates the CAN-12 has strong preferentialexpression in esophagus, lymph node, spinal cord, and to a lesserextent, in thymus, and spleen. Based on this information, we haveprovisionally named the gene and protein CAN-12.

In the present invention, “isolated” refers to material removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring), and thus is altered “by the hand of man” from its naturalstate. For example, an isolated polynucleotide could be part of a vectoror a composition of matter, or could be contained within a cell, andstill be “isolated” because that vector, composition of matter, orparticular cell is not the original environment of the polynucleotide.The term “isolated” does not refer to genomic or cDNA libraries, wholecell total or mRNA preparations, genomic DNA preparations (includingthose separated by electrophoresis and transferred onto blots), shearedwhole cell genomic DNA preparations or other compositions where the artdemonstrates no distinguishing features of the polynucleotide/sequencesof the present invention.

In specific embodiments, the polynucleotides of the invention are atleast 15, at least 30, at least 50, at least 100, at least 125, at least500, or at least 1000 continuous nucleotides but are less than or equalto 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb,2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides ofthe invention comprise a portion of the coding sequences, as disclosedherein, but do not comprise all or a portion of any intron. In anotherembodiment, the polynucleotides comprising coding sequences do notcontain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ tothe gene of interest in the genome). In other embodiments, thepolynucleotides of the invention do not contain the coding sequence ofmore than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1genomic flanking gene(s).

As used herein, a “polynucleotide” refers to a molecule having a nucleicacid sequence contained in SEQ ID NO:1, SEQ ID NO:53, SEQ ID NO:55, SEQID NO:23 or the cDNA contained within the clone(s) deposited with theATCC. For example, the polynucleotide can contain the nucleotidesequence of the full length cDNA sequence, including the 5′ and 3′untranslated sequences, the coding region, with or without a signalsequence, the secreted protein coding region, as well as fragments,epitopes, domains, and variants of the nucleic acid sequence. Moreover,as used herein, a “polypeptide” refers to a molecule having thetranslated amino acid sequence generated from the polynucleotide asbroadly defined.

In the present invention, the full length sequence identified as SEQ IDNO:1, SEQ ID NO:53, SEQ ID NO:55, and SEQ ID NO:23 was often generatedby overlapping sequences contained in one or more clones (contiganalysis). A representative clone containing all or most of the sequencefor SEQ ID NO:1, SEQ ID NO:53, SEQ ID NO:55, and/or, SEQ ID NO:23 wasdeposited with the American Type Culture Collection (“ATCC”). As shownin Table I, each clone is identified by a cDNA Clone ID (Identifier) andthe ATCC Deposit Number. The ATCC is located at 10801 UniversityBoulevard, Manassas, Va., 20110-2209, USA. The ATCC deposit was madepursuant to the terms of the Budapest Treaty on the internationalrecognition of the deposit of microorganisms for purposes of patentprocedure. The deposited clone is inserted in the pSport1 plasmid (LifeTechnologies) using the NotI and SalI restriction endonuclease cleavagesites.

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373, preferably a Model 3700, from AppliedBiosystems, Inc.), and all amino acid sequences of polypeptides encodedby DNA molecules determined herein were predicted by translation of aDNA sequence determined above. Therefore, as is known in the art for anyDNA sequence determined by this automated approach, any nucleotidesequence determined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art. As is also known inthe art, a single insertion or deletion in a determined nucleotidesequence compared to the actual sequence will cause a frame shift intranslation of the nucleotide sequence such that the predicted aminoacid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

Using the information provided herein, such as the nucleotide sequencein FIGS. 1A-E (SEQ ID NO:1), a nucleic acid molecule of the presentinvention encoding the CAN-12 polypeptide may be obtained using standardcloning and screening procedures, such as those for cloning cDNAs usingmRNA as starting material. Illustrative of the invention, the nucleicacid molecule described in FIGS. 1A-E (SEQ ID NO:1) was discovered in acDNA library derived from human liver, brain and testis and spleen.

The determined nucleotide sequence of the CAN-12 cDNA in FIGS. 1A-E (SEQID NO:1) contains an open reading frame encoding a protein of about 428amino acid residues, with a deduced molecular weight of about 49.5 kDa.The amino acid sequence of the predicted CAN-12 polypeptide is shown inFIGS. 1A-E (SEQ ID NO:24).

Using the information provided herein, such as the nucleotide sequencein FIGS. 8A-C (SEQ ID NO:53), a nucleic acid molecule of the presentinvention encoding the CAN-12v1 polypeptide may be obtained usingstandard cloning and screening procedures, such as those for cloningcDNAs using mRNA as starting material. Illustrative of the invention,the nucleic acid molecule described in FIGS. 8A-C (SEQ ID NO:53) wasdiscovered in a cDNA library derived from human liver, brain and testisand spleen.

The determined nucleotide sequence of the CAN-12v1 cDNA in FIGS. 8A-C(SEQ ID NO:53) contains an open reading frame encoding a protein ofabout 694 amino acid residues, with a deduced molecular weight of about80.3 kDa. The amino acid sequence of the predicted CAN-12v1 polypeptideis shown in FIGS. 8A-C (SEQ ID NO:54).

Using the information provided herein, such as the nucleotide sequencein FIGS. 9A-C (SEQ ID NO:55), a nucleic acid molecule of the presentinvention encoding the CAN-12v2 polypeptide may be obtained usingstandard cloning and screening procedures, such as those for cloningcDNAs using mRNA as starting material. Illustrative of the invention,the nucleic acid molecule described in FIGS. 9A-C (SEQ ID NO:55) wasdiscovered in a cDNA library derived from human liver, brain and testisand spleen.

The determined nucleotide sequence of the CAN-12v2 cDNA in FIGS. 9A-C(SEQ ID NO:55) contains an open reading frame encoding a protein ofabout 697 amino acid residues, with a deduced molecular weight of about80.6 kDa. The amino acid sequence of the predicted CAN-12v2 polypeptideis shown in FIGS. 9A-C (SEQ ID NO:56).

A “polynucleotide” of the present invention also includes thosepolynucleotides capable of hybridizing, under stringent hybridizationconditions, to sequences contained in SEQ ID NO:1, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:23, the complement thereof, or the cDNA within theclone deposited with the ATCC. “Stringent hybridization conditions”refers to an overnight incubation at 42 degree C. in a solutioncomprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed bywashing the filters in 0.1×SSC at about 65 degree C.

Also contemplated are nucleic acid molecules that hybridize to thepolynucleotides of the present invention at lower stringencyhybridization conditions. Changes in the stringency of hybridization andsignal detection are primarily accomplished through the manipulation offormamide concentration (lower percentages of formamide result inlowered stringency); salt conditions, or temperature. For example, lowerstringency conditions include an overnight incubation at 37 degree C. ina solution comprising 6×SSPE (20×SSPE 3M NaCl; 0.2M NaH2PO4; 0.02M EDTA,pH 7.4), 0.5% SDS, 30% formamide, 100 μg/mi salmon sperm blocking DNA;followed by washes at 50 degree C. with 1×SSPE, 0.1% SDS. In addition,to achieve even lower stringency, washes performed following stringenthybridization can be done at higher salt concentrations (e.g. 5×SSC).

Note that variations in the above conditions may be accomplished throughthe inclusion and/or substitution of alternate blocking reagents used tosuppress background in hybridization experiments. Typical blockingreagents include Denhardt's reagent, BLOTTO, heparin, denatured salmonsperm DNA, and commercially available proprietary formulations. Theinclusion of specific blocking reagents may require modification of thehybridization conditions described above, due to problems withcompatibility.

Of course, a polynucleotide which hybridizes only to polyA+ sequences(such as any 3′ terminal polyA+ tract of a cDNA shown in the sequencelisting), or to a complementary stretch of T (or U) residues, would notbe included in the definition of “polynucleotide,” since such apolynucleotide would hybridize to any nucleic acid molecule containing apoly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone generated using oligo dT as a primer).

The polynucleotide of the present invention can be composed of anypolyribonucleotide or polydeoxribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. For example, polynucleotides can becomposed of single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, the polynucleotide can be composed oftriple-stranded regions comprising RNA or DNA or both RNA and DNA. Apolynucleotide may also contain one or more modified bases or DNA or RNAbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

The polypeptide of the present invention can be composed of amino acidsjoined to each other by peptide bonds or modified peptide bonds, i.e.,peptide isosteres, and may contain amino acids other than the 20gene-encoded amino acids. The polypeptides may be modified by eithernatural processes, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic polypeptides may result fromposttranslation natural processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, Proteins—Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993); PosttranslationalCovalent Modification of Proteins, B. C. Johnson, Ed., Academic Press,New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646(1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)

“A polypeptide having biological activity” refers to polypeptidesexhibiting activity similar, but not necessarily identical to, anactivity of a polypeptide of the present invention, including matureforms, as measured in a particular biological assay, with or withoutdose dependency. In the case where dose dependency does exist, it neednot be identical to that of the polypeptide, but rather substantiallysimilar to the dose-dependence in a given activity as compared to thepolypeptide of the present invention (i.e., the candidate polypeptidewill exhibit greater activity or not more than about 25-fold less and,preferably, not more than about tenfold less activity, and mostpreferably, not more than about three-fold less activity relative to thepolypeptide of the present invention.)

The term “organism” as referred to herein is meant to encompass anyorganism referenced herein, though preferably to eukaryotic organisms,more preferably to mammals, and most preferably to humans.

As used herein the terms “modulate” or “modulates” refer to an increaseor decrease in the amount, quality or effect of a particular activity,DNA, RNA, or protein. The definition of “modulate” or “modulates” asused herein is meant to encompass agonists and/or antagonists of aparticular activity, DNA, RNA, or protein.

The present invention encompasses the identification of proteins,nucleic acids, or other molecules, that bind to polypeptides andpolynucleotides of the present invention (for example, in areceptor-ligand interaction). The polynucleotides of the presentinvention can also be used in interaction trap assays (such as, forexample, that described by Ozenberger and Young (Mol Endocrinol.,9(10):1321-9, (1995); and Ann. N. Y. Acad. Sci., 7;766:279-81, (1995)).

The polynucleotide and polypeptides of the present invention are usefulas probes for the identification and isolation of full-length cDNAsand/or genomic DNA which correspond to the polynucleotides of thepresent invention, as probes to hybridize and discover novel, relatedDNA sequences, as probes for positional cloning of this or a relatedsequence, as probe to “subtract-out” known sequences in the process ofdiscovering other novel polynucleotides, as probes to quantify geneexpression, and as probes for microarrays.

In addition, polynucleotides and polypeptides of the present inventionmay comprise one, two, three, four, five, six, seven, eight, or moremembrane domains.

Also, in preferred embodiments the present invention provides methodsfor further refining the biological function of the polynucleotidesand/or polypeptides of the present invention.

Specifically, the invention provides methods for using thepolynucleotides and polypeptides of the invention to identify orthologs,homologs, paralogs, variants, and/or allelic variants of the invention.Also provided are methods of using the polynucleotides and polypeptidesof the invention to identify the entire coding region of the invention,non-coding regions of the invention, regulatory sequences of theinvention, and secreted, mature, pro-, prepro-, forms of the invention(as applicable).

In preferred embodiments, the invention provides methods for identifyingthe glycosylation sites inherent in the polynucleotides and polypeptidesof the invention, and the subsequent alteration, deletion, and/oraddition of said sites for a number of desirable characteristics whichinclude, but are not limited to, augmentation of protein folding,inhibition of protein aggregation, regulation of intracellulartrafficking to organelles, increasing resistance to proteolysis,modulation of protein antigenicity, and mediation of intercellularadhesion.

In further preferred embodiments, methods are provided for evolving thepolynucleotides and polypeptides of the present invention usingmolecular evolution techniques in an effort to create and identify novelvariants with desired structural, functional, and/or physicalcharacteristics.

The present invention further provides for other experimental methodsand procedures currently available to derive functional assignments.These procedures include but are not limited to spotting of clones onarrays, micro-array technology, PCR based methods (e.g., quantitativePCR), anti-sense methodology, gene knockout experiments, and otherprocedures that could use sequence information from clones to build aprimer or a hybrid partner.

Polynucleotides and Polvpeptides of the Invention

Features of the Polypeptide Encoded by Gene No:1

The polypeptide of this gene provided as SEQ ID NO:24 (FIGS. 1A-E),encoded by the polynucleotide sequence according to SEQ ID NO:1 (FIGS.1A-E), and/or encoded by the polynucleotide contained within thedeposited clone, CAN-12, has significant homology at the nucleotide andamino acid level to a number of calpains, which include, for example,the human CAN10 protein (type II diabetes linked) (hCAN10; GenbankAccession No: gi|NP_(—)075574; SEQ ID NO:3); the human CAN5 protein(hCAN5; Genbank Accession No: gi|NP_(—)004046; SEQ ID NO:4); the largecatalytic subunit of the human CALPAIN 1 protein (also referred to asCalcium-Activated Neutral Proteinase, CANP, μ-TYPE) (HCAN1; GenbankAccession No: gi|12408656; SEQ ID NO:5); the large subunit of the humancalpain 3 protein (EC 3.4.22.17) (also referred to as CALPAIN L3,CALPAIN P94, Calcium-Activated Neutral Proteinase 3, CANP 3;muscle-specific calcium-activated neutral protease 3 large subunit)(hCAN3; Genbank Accession No: gi|4557405; SEQ ID NO:6); the largecatalytic subunit of the mouse CALPAIN 1 protein (also referred to asCalcium-Activated Neutral Proteinase) (CANP) μ-TYPE ) (mCALPAIN1;Genbank Accession No: gi|088666; SEQ ID NO:7); the mouse CALPAIN LP82(nLP82; Genbank Accession No: gi|3661585; SEQ ID NO:8); the ratstomach-specific calcium-activated neutral protease large subunit, NCL2protein (rNCL2; Genbank Accession No: gi|NP_(—)006606; SEQ ID NO:9); thehuman CAN11 protein (hCAN11; Genbank Accession No: gi|NP-008989; SEQ IDNO:10); the human CAN2 protein (hCAN2; Genbank Accession No: gi|4502563;SEQ ID NO:11); and the human CAN9 protein (hCAN9; Genbank Accession No:gi|5729758; SEQ ID NO:12). An alignment of the CAN-12 polypeptide withthese proteins is provided in FIGS. 2A-E. Based upon such strongconservation, the inventors have ascribed the CAN-12 polypeptide ashaving proteolytic activity, preferably calpain activity.

The CAN-12+ (SEQ ID NO:2) polypeptide was determined to have 30.7%identity and 38.0% similarity with the human CAN10 protein (type IIdiabetes linked) (hCAN10; Genbank Accession No: gi|NP_(—)075574; SEQ IDNO:3); to have 34.2% identity and 45.4% similarity with the human CAN5protein (hCAN5; Genbank Accession No: gi|NP_(—)004046; SEQ ID NO:4); tohave 37.9% identity and 47.4% similarity with the large catalyticsubunit of the human CALPAIN 1 protein (also referred to asCalcium-Activated Neutral Proteinase, CANP, μ-TYPE) (hCAN1; GenbankAccession No: gi|12408656; SEQ ID NO:5); to have 36.3% identity and43.6% similarity with the large subunit of the human calpain 3 protein(EC 3.4.22.17) (also referred to as CALPAIN L3, CALPAIN P94,Calcium-Activated Neutral Proteinase 3, CANP 3; muscle-specificcalcium-activated neutral protease 3 large subunit) (hCAN3; GenbankAccession No: gi|4557405; SEQ ID NO:6); to have 39.0% identity and 47.6%similarity with the large catalytic subunit of the mouse CALPAIN 1protein (also referred to as Calcium-Activated Neutral Proteinase)(CANP) μ-TYPE ) (mCALPAIN1; Genbank Accession No: gi|O88666; SEQ IDNO:7); to have 37.8% identity and 45.3% similarity with the mouseCALPAIN LP82 (mLP82; Genbank Accession No: gi|3661585; SEQ ID NO:8); tohave 34.2% identity and 45.4% similarity with the rat stomach-specificcalcium-activated neutral protease large subunit, NCL2 protein (rNCL2;Genbank Accession No: gi|NP_(—)006606; SEQ ID NO:9); to have 40.4%identity and 47.3% similarity with the human CAN11 protein (HCAN11;Genbank Accession No: gi|NP_(—)008989; SEQ ID NO:10); to have 36.8%identity and 45.8% similarity with the human CAN2 protein (hCAN2;Genbank Accession No: gi|4502563; SEQ ID NO:11); and to have 39.4%identity and 47.2% similarity with the human CAN9 protein (hCAN9;Genbank Accession No: gi|5729758; SEQ ID NO:12). The polypeptidesequence used for these percent identity and similarity values comprisedthe additional amino acids that extend beyond amino acid 428 of SEQ IDNO:24—specifically, SEQ ID NO:2.

The CAN-12 polypeptide was determined to have 34.3% identity and 42.3%similarity with the human CAN10 protein (type II diabetes linked)(hCAN10; Genbank Accession No: gi|NP_(—)075574; SEQ ID NO:3); to have40.5% identity and 51.9% similarity with the human CAN5 protein (hCAN5;Genbank Accession No: gi|NP_(—)004046; SEQ ID NO:4); to have 44.3%identity and 51.9% similarity with the large catalytic subunit of thehuman CALPAIN 1 protein (also referred to as Calcium-Activated NeutralProteinase, CANP, μ-TYPE) (hCAN1; Genbank Accession No: gi|12408656; SEQID NO:5); to have 44.9% identity and 51.8% similarity with the largesubunit of the human calpain 3 protein (EC 3.4.22.17) (also referred toas CALPAIN L3, CALPAIN P94, Calcium-Activated Neutral Proteinase 3, CANP3; muscle-specific calcium-activated neutral protease 3 large subunit)(hCAN3; Genbank Accession No: gi|4557405; SEQ ID NO:6); to have 46.1%identity and 52.7% similarity with the large catalytic subunit of themouse CALPAIN 1 protein (also referred to as Calcium-Activated NeutralProteinase) (CANP) μ-TYPE ) (mCALPAIN1; Genbank Accession No: gi|O88666;SEQ ID NO:7); to have 46.2% identity and 53.5% similarity with the mouseCALPAIN LP82 (mLP82; Genbank Accession No: gi|3661585; SEQ ID NO:8); tohave 44.6% identity and 51.4% similarity with the rat stomach-specificcalcium-activated neutral protease large subunit, NCL2 protein (rNCL2;Genbank Accession No: gi|NP_(—)006606; SEQ ID NO:9); to have 45.5%identity and 51.7% similarity with the human CAN11 protein (hCAN11;Genbank Accession No: gi|NP_(—)008989; SEQ ID NO:10); to have 46.2%identity and 53.5% similarity with the human CAN2 protein (hCAN2;Genbank Accession No: gi|4502563; SEQ ID NO:11); and to have 44.3%identity and 51.9% similarity with the human CAN9 protein (hCAN9;Genbank Accession No: gi|5729758; SEQ ID NO:12). The polypeptidesequence used for these percent identity and similarity values was thefull-length CAN-12 polypeptide (SEQ ID NO:24).

The human CAN10 protein (type II diabetes linked) (HCAN10; GenbankAccession No: gi|NP_(—)075574; SEQ ID NO:3) is a human calpain gene thatencodes a large calpain subunit. CAN10 is an atypical calpain in that itlacks the calmodulin-like calcium-binding domain and instead has adivergent C-terminal domain. CAN10 is similar in organization tocalpains 5 and 6 and is associated with type 2 or non-insulin-dependentdiabetes mellitus (NIDDM) and located within the NIDDM1 chromosomalregion (Nat. Genet. 26 (2), 163-175 (2000)).

The large subunit of the human calpain 3 protein (EC 3.4.22.17) (alsoreferred to as CALPAIN L3, CALPAIN P94, Calcium-Activated NeutralProteinase 3, CANP 3; muscle-specific calcium-activated neutral protease3 large subunit) (hCAN3; Genbank Accession No: gi|4557405; SEQ ID NO:6)is a muscle-specific member of the calpain large subunit family. Loss ofCAPN3 function has been associated with limb-girdle muscular dystrophiestype 2A (Cell 81 (1), 2740 (1995)).

The human CAN9 protein (hCAN9; Genbank Accession No: gi|5729758; SEQ IDNO:12) is a calpain that is expressed predominantly in stomach and smallintestine and is thought to have specialized functions in the digestivetract, and be associated with gastric cancer. (Biol. Chem. 379 (2),175-183 (1998); and Jpn. J. Cancer Res. 91 (5), 459-463 (2000)).

As described above, the CAN-12 polypeptide was found to have significantsequence homology with calpains, particularly members of the m-calpainfamily. A conserved peptide signature ofQx3(G,E)xC(Y,W)x2(S,T,A,G,C)(S,T,A,G,C,V) Qx{3}(G)xC(W)x{2}(A)(A)(referred to as a thiol (cysteine) protease active site domain) commonto most calpain family members is found in the protein sequence ofCAN-12 from amino acid 90 to amino acid 111 of SEQ ID NO:24 (FIGS.1A-E). Protein threading and molecular modeling of CAN-12 suggests thatCAN-12 has a structural fold similar to representative m-calpains.Moreover, the structural and threading alignments of the presentinvention suggest that amino acids 101 (“C”), 253 (“H”), and 277 (“N”)of SEQ ID NO:24 (FIGS. 1A-E) may represent the catalytic amino acidswithin the active site domain. Thus, based upon the sequence andstructural homology to known calpains, particularly the presence of thethiol cysteine protease active site domain, the novel CAN-12 is believedto represent a novel human calpain.

In an alternative embodiment, the following polypeptide is encompassedby the present invention:MSLWPPFRCRWKLAPRYSRRASPQQPQQDFEALLAECLRNGCLFEDTSFPATLSSIGSGSLLQKLPPRLQWKRPPELHSNPQFYFARRRRLDLCQGIVGDCWFLAALQALALHQDILSRVVPLNQSFTEKYAGIFRFWFWHYGNWVPVVIDDRLPVNEAGQLVFVSSTYKNLFWGALLEKAYAKLSGSYEDLQSGQVSEALVDFTGGVTMTINLAEAHGNLWDILIEATYNRTLIGCQTHSGKILENGLVEGHAYTLTGIRKVTCKHRPEYLVKLRNPWGKVEWKGDWSDSSSKWELLSPKEKILLLRKDNDGEFWMTLQDFKTHFVLLVICKLTPGLLSQEAAQKWTYTMREGRWEKRSTAGGQRQLLQDTFWKNPQFLLSVWRPEEGRRSLRPCSVLVSLLQKPRHRCRKRKPLLAIGFYLYRMNK (SEQ ID NO:24). Polynucleotidesencoding these polypeptides are also provided (SEQ ID NO:23).

In confirmation of the strong homology to known calpains, the CAN-12polypeptide was determined to have several conserved catalytic aminoacids at amino acid C101, H253, and N277 of SEQ ID NO:24 (FIGS. 1A-E).As discussed more particularly herein, calpains are a group ofstructurally diverse, high molecular weight (400 to 500 amino acids)proteins that have a catalytic cysteine amino acid and one or morecalcium binding domains. Despite the structural heterogeneity, calpainsshare some well defmed structural-functional characteristics,particularly in their active site domains.

In preferred embodiments, the CAN-12 polypeptide of the presentinvention is directed to a polypeptide having structural similarity tocalpains.

Based upon the strong homology to members of the calpain family, theCAN-12 polypeptide is expected to share at least some biologicalactivity with calpains, preferably with m-calpain family members, andmore preferable to the large subunits of m-calpain family members, inaddition to other calpains and calpain subunits referenced herein and/orotherwise known in the art.

Expression profiling designed to measure the steady state mRNA levelsencoding the CAN-12 polypeptide showed predominately high expressionlevels in spinal cord tissue; significantly high expression in lymphnode and thymus, and to a lesser extent, in spleen tissue (See FIG. 4).

Expanded analysis of CAN-12 expression levels by TaqMan™ quantitativePCR (see FIG. 6) confirmed that the CAN-12 polypeptide is expressed inthe lymph gland. However, the TaqMan™ quantitative PCR determined thatthe CAN-12 polypeptide is primarily expressed in the eosophagus. Infact, with the exception of the lymph gland, the steady state mRNA levelof CAN-12 was approximately 2700 times higher in the esophagus than inall other tissues tested. These data suggest modulators of the CAN-12polynucleotides and polypeptides may be useful for the treatment,detection, and/or amelioration of the following, non-limiting diseasesand disorders associated with the eosphagus: dysphagia, cricoharyngealincoordination, esophageal carcinoma, esophageal webs, achalasia,symptomatic diffuse esophageal spasm, gastroesophageal reflux, and/orcorrosive esophagitis.

The polynucleotides encoding the CAN-12 polypeptide of the presentinvention were used to determine the chromosomal localization of thecalpain12 gene. Polynuclelotides corresponding to CAN-12 (SEQ IID NO:1)were shown to localize to chromosome 2, specifically 2p16-p21. Thecomparison of the chromosomal location of the calpain12 gene with thelocation of chromosomal regions which have been shown to be associatedwith specific diseases or conditions, e.g. by linkage analysis, can beindicative of diseases in which calpain12 may play a role.Interestingly, a whole-genome linkage scan in multiple sclerosisfamilies (Ebers et al. A full genome search in multiple sclerosis.Nature Genet. 13: 472-476, 1996.) identified 5 susceptibility loci onchromosomes 2, 3, 5, 11, and X. In particular, an association wasidentified with marker D2S119 on chromosome 2 and MS. The localizationof the D2S119 marker was further delimeated to 2p16-p21 based on aradiation hybrid linkage map retrieved from an online query at NCBI website: http:/www.ncbi.nlm.nih.gov/genome/sts/. Since the map of calpain12 and the susceptibility marker D2S119 overlaps, it is reasonable topostulate that calpain 12 may contribute to MS. Furthermore, thetranscription profile of calpain12 indicated a prominent expression inspinal cord, and implication of calpains in MS has been suggested(Shields D C et al. A putative mechanism of demyelination in multiplesclerosis by a proteolytic enzyme, calpain. Proc Natl Acad Sci USA.96:11486-91.1999).

The CAN-12 polynucleotides and polypeptides of the present invention,including agonists, antagonists, and/or fragments thereof, have usesthat include modulating cellular adhesion events, cellularproliferation, and inflammation, in various cells, tissues, andorganisms, and particularly in mammalian spinal cord tissue, lymph node,thymus, and spleen tissue, preferably human tissue. CAN-12polynucleotides and polypeptides of the present invention, includingagonists and/or fragments thereof, may-be useful in diagnosing,treating, prognosing, and/or preventing neural, immune, hematopoietic,and/or proliferative diseases or disorders.

The strong homology to human calpains, particularly m-calpains, combinedwith the predominate localized expression in esophagus tissue suggeststhe CAN-12 polynucleotides and polypeptides may be useful in treating,diagnosing, prognosing, and/or preventing gastrointestinal diseases,particularly esophageal diseases and/or disorders which include thefollowing non-limiting examples: aberrant transport of food bolus fromthe mouth to the stomach, aberrant prevention of retrograde flow ofgastrointestinal contents, aberrant esophageal peristaltic contractions,pyrosis, painful swallowing, reflux esophagitis, esophageal motilitydisorders, esophageal spasms, diffuse esophageal spasm, atypical chestpain, regurgitation, oropharyngeal paralysis, nasal regurgitation,dysphagia, cricopharyngeal bar, globus pharyngeus, achalasia, motordisorders of the esophageal smooth muscle, scleroderma esophagus,gastroesophageal reflux disease (GERD), esophagitis, Barrett'sesophagus, viral esophagitis, Herpes simplex virus mediated viralesophagitis, Varicella-zoster virus mediated viral esophagitis,Cytomegalovirus mediated viral esophagitis, bacterial esophagitis,Lactobacillus mediated bacterial esophagitis, Candida mediatedesophagitis, radiation esophagitis, corrosive esophagitis, pill-inducedesophagitis, esophagitis associated with mucocutaneous and systemicdiseases, diverticula, lower esophageal mucosal ring, lower esophagealmuscular ring, hiatal hernia, paraesophageal hernia, esophageal rupture,and/or Mallory-Weiss Syndrome.

Although calpains are typically associated primarily withneurogenerative conditions, their association in gastrointenstinaltissues has precedence. For example, the human CAN9 protein (hCAN9;Genbank Accession No: gi|5729758; SEQ ID NO:12) is predominatelyexpressed in the stomach and small intestine and is thought to beassociated with gastric cancers.

The strong homology to human calpains, particularly m-calpains, combinedwith the predominate expression in spinal cord tissue suggests theCAN-12 polynucleotides and polypeptides may be useful in treating,diagnosing, prognosing, and/or preventing neural diseases,neurodegenerative disease states, behavioral disorders, or inflammatoryconditions. Representative uses are described in the “NeurologicalDiseases”, “Regeneration” and “Hyperproliferative Disorders” sectionsbelow, in the Examples, and elsewhere herein. Briefly, the uses include,but are not limited to the detection, treatment, and/or prevention ofAlzheimer's Disease, Parkinson's Disease, Huntington's Disease, TouretteSyndrome, meningitis, encephalitis, demyelinating diseases, peripheralneuropathies, neoplasia, trauma, congenital malformations, spinal cordinjuries, ischemia and infarction, aneurysms, hemorrhages,schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder,depression, panic disorder, learning disabilities, ALS, psychoses,autism, and altered behaviors, including disorders in feeding, sleeppatterns, balance, and perception. In addition, elevated expression ofthis gene product in regions of the brain indicates it plays a role innormal neural function. Potentially, this gene product is involved insynapse formation, neurotransmission, learning, cognition, homeostasis,or neuronal differentiation or survival. Furthermore, the protein mayalso be used to determine biological activity, to raise antibodies, astissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

Alternatively, the strong homology to human calpains, particularlym-calpains, combined with the localized expression in lymph node,thymus, and spleen tissue suggests the CAN-12 polynucleotides andpolypeptides may be useful in treating, diagnosing, prognosing,ameliorating, and/or preventing immune diseases and/or disorders.Representative uses are described in the “Immune Activity” and“Infectious Disease” sections below, and elsewhere herein. Briefly, thestrong expression in immune tissue indicates a role in regulating theproliferation; survival; differentiation; and/or activation ofhematopoietic cell lineages, including blood stem cells. The CAN-12polypeptide may also be useful as a preventative agent for immunologicaldisorders including arthritis, asthma, immunodeficiency diseases such asAIDS, leukemia, rheumatoid arthritis, granulomatous disease,inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia,psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity;immune reactions to transplanted organs and tissues, such ashost-versus-graft and graft-versus-host diseases, or autoimmunitydisorders, such as autoimmune infertility, lense tissue injury,demyelination, systemic lupus erythematosis, drug induced hemolyticanemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.Moreover, the protein may represent a secreted factor that influencesthe differentiation or behavior of other blood cells, or that recruitshematopoietic cells to sites of injury. Thus, this gene product may beuseful in the expansion of stem cells and committed progenitors ofvarious blood lineages, and in the differentiation and/or proliferationof various cell types.

Moreover, the protein would be useful in the detection, treatment,and/or prevention of a variety of vascular disorders and conditions,which include, but are not limited to miscrovascular disease, vascularleak syndrome, aneurysm, stroke, embolism, thrombosis, coronary arterydisease, arteriosclerosis, and/or atherosclerosis. Furthermore, theprotein may also be used to determine biological activity, raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

In addition, antagonists of the CAN-12 polynucleotides and polypeptidesmay have uses that include diagnosing, treating, prognosing, and/orpreventing diseases or disorders related to hyper calpain activity,which may include immune and/or proliferative diseases or disorders,particularly thrombosis, embolism, and other blood disorders.Therapeutic and/or pharmaceutical compositions comprising the CAN-12polypeptides may be formulated to comprise heparin.

In addition, antagonists of the CAN-12 polynucleotides and polypeptidesmay have uses that include diagnosing, treating, ameliorating,prognosing, and/or preventing diseases or disorders related to hypercalpain activity, which may include neuronal excitotoxicity, ischemicstroke, hemoragic stroke, hypoxic stress, trauma, cell destruction,spinal cord injury following trauma, degeneration of vulnerablehippocampal neurons after ischemia, reovirus-induced apoptosis,viral-induced induced myocarditis, acute and chronic inflammation,cataract formation, multiple sclerosis, demylenating disorders, acoustictrauma, hearing loss caused by noise, neuronal damage, cardiac ischemicdamage, and/or hepatocyte necrosis during and following anoxia

CAN-12 polynucleotides and polypeptides, including fragments and/orantagonsists thereof, may have uses which include modulatingdevelopment, differentiation, cellular transformation in response tocell signaling, cell-cell and/or cell-extracellular matrix interactions,clustering of the integrin receptor aIIb3, modulating in long termpotentiation (memory), modulating neurite outgrowth, modulating corticallamination activation of protein kinases and phosphatases, remodelingand disassembling the cytoskeleton, cell cycle modulation, in addition,to ameliorating, preventing, and/or treating limb-girdle musculardystrophy (LGMD), insulin resistance in diabetics, Alzheimer's disease,Multiple sclerosis, Huntington's disease, Parkinson's disease andamyotrophy.

Moreover, CAN-12 polynucleotides and polypeptides, including fragmentsand agonists thereof, may have uses which include treating, diagnosing,prognosing, and/or preventing hyperproliferative disorders, particularlyof the neural and immune systems. Such disorders may include, forexample, cancers, and metastatic conditions.

CAN-12 polynucleotides and polypeptides, including fragments and/orantagonsists thereof, may have uses which include identification ofmodulators of CAN-12 function including antibodies (for detection orneutralization), naturally-occurring modulators and small moleculemodulators. Antibodies to domains (including CAN-12 epitopes providedherein) of the CAN-12 protein could be used as diagnostic agents ofinflammatory conditions in patients, are useful in monitoring theactivation and presence of cognate proteases, and can be used as abiomarker for the protease involvement in disease states and in theevaluation of inhibitors of the cognate protease in vivo.

CAN-12 polypeptides and polynucleotides are useful for diagnosingdiseases related to over or under expression of CAN-12 proteins byidentifying mutations in the CAN-12 gene using CAN-12 probes, ordetermining CAN-12 protein or mRNA expression levels. CAN-12polypeptides are also useful for screening for compounds, which affectactivity of the protein. Diseases that can be treated with CAN-12include, the following, non-limiting examples: neuro-regeneration,neuropathic pain, obesity, anorexia, HIV infections, cancers, bulimia,asthma, Parkinson's disease, acute heart failure, hypotension,hypertension, osteoporosis, angina pectoris, myocardial infarction,psychotic, immune, metabolic, cardiovascular, and neurologicaldisorders.

The predominate expression in neural tissues, combined with thesignificant expression in a number of other tissues, suggests the CAN-12polynucleotide and polypeptide of the present invention may be involvedin modulating nerve invasion, innervation, nerve maintenance, andpotentially myeline sheath maintenance and integrity.

The CAN-12 polynucleotides and polypeptides, including fragments andantagonists thereof, may have uses which include detecting, diagnosing,treating, ameliorating, and/or preventing diseases and disorders of theneural system, particularly Alzheimer's disease, either directly orindirectly, in addition to other neural disorders known in the art orprovided in the “Neurological Diseases” section herein, such asmodulating nerve invasion, innervation, nerve maintenance, potentiallymyelin sheath maintenance and integrity, encephalomyelitis, autoimmuneencephalomyelitis, human T cell leukemia virus type I(HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP),and neuro-inflammatory diseases.

Molecular genetic manipulation of the structure of the active sitedomain, particularly the predicted catalytic amino acids, and of otherfunctional domains in the calpain family (e.g., active site domainbinding pocket) enables the production of calpains with tailor-madeactivities. Thus, the CAN-12 polypeptides, and fragments thereof, aswell as any homologous product resulting from genetic manipulation ofthe structure, are useful for NMR-based design of modulators of CAN-12biological activity, and calpains, in general.

CAN-12 polypeptides and polynucleotides have additional uses whichinclude diagnosing diseases related to the over and/or under expressionof CAN-12 by identifying mutations in the CAN-12 gene by using CAN-12sequences as probes or by determining CAN-12 protein or mRNA expressionlevels. CAN-12 polypeptides may be useful for screening compounds thataffect the activity of the protein. CAN-12 peptides can also be used forthe generation of specific antibodies and as bait in yeast two hybridscreens to find proteins the specifically interact with CAN-12(described elsewhere herein).

The CAN-12 polynucleotides and polypeptides, including fragments andagonists thereof, may have uses which include detecting, diagnosing,treating, ameliorating, and/or preventing metabolic diseases anddisorders, such as diabetes. Moreover, expressed human CAN-12 may beuseful in the detection of patients susceptible to diabetes. Alsoparadigms that would simulate intracellular CAN-12 activity would beuseful in treating diabetes.

The CAN-12 polynucleotides and polypeptides, including fragmentsthereof, may have uses which include identifying inhibitors ofintracellular calpain inhibitors (calpastatins) leading to an effectiveincrease in calpain activity.

Various approaches to detect alterations or allelic variants at thegenomic or mRNA level of CAN-12, could be used as a diagnostic foridentifying MS patients, or individuals susceptible to have MS. It islikely that the calpain12 gene comprises polymorphic sites (i.e. SNPs),with specific alleles which may be associated with MS or otherneurodegenerative disorders, or associated with an increased likelihoodof developing these diseases. Therefore, the invention provides thecalpain 12 sequence that can be used to design specific primers for theidentification of polymorphisms or mutations in calpain12 of patientsaffected with MS. The presence of a specific allele variant, such as aSNP allele or SNPs haplotype that renders the subject carrying it moresusceptible to develop MS or other related diseases could be identified(e.g. a variant in the can12 promoter region that increased transcriptlevels of can12, or mutations in the coding sequence that increased thestability or half-life of the can12 protein). Other methods such asNorthern-blot analysis could be performed to measure transcript levelsusing a can12 cDNA probe derived from the sequence of the invention.

Although it is believed the encoded polypeptide may share at least somebiological activities with human calpains (particularly m-calpains), anumber of methods of determining the exact biological function of thisclone are either known in the art or are described elsewhere herein.Briefly, the function of this clone may be determined by applyingmicroarray methodology. Nucleic acids corresponding to the CAN-12polynucleotides, in addition to, other clones of the present invention,may be arrayed on microchips for expression profiling. Depending onwhich polynucleotide probe is used to hybridize to the slides, a changein expression of a specific gene may provide additional insight into thefunction of this gene based upon the conditions being studied. Forexample, an observed increase or decrease in expression levels when thepolynucleotide probe used comes from diseased neural tissue, as comparedto, normal tissue might indicate a function in modulating neuralfunction, for example. In the case of CAN-12, spinal cord, lymph node,thymus, and/or spleen tissue should be used to extract RNA to preparethe probe.

In addition, the function of the protein may be assessed by applyingquantitative PCR methodology, for example. Real time quantitative PCRwould provide the capability of following the expression of the CAN-12gene throughout development, for example. Quantitative PCR methodologyrequires only a nominal amount of tissue from each developmentallyimportant step is needed to perform such experiments. Therefore, theapplication of quantitative PCR methodology to refining the biologicalfunction of this polypeptide is encompassed by the present invention. Inthe case of CAN-12, a disease correlation related to CAN-12 may be madeby comparing the mRNA expression level of CAN-12 in normal tissue, ascompared to diseased tissue (particularly diseased tissue isolated fromthe following: esophagus, spinal cord, lymph node, thymus, and/or spleentissue). Significantly higher or lower levels of CAN-12 expression inthe diseased tissue may suggest CAN-12 plays a role in diseaseprogression, and antagonists against CAN-12 polypeptides would be usefultherapeutically in treating, preventing, and/or ameliorating thedisease. Alternatively, significantly higher or lower levels of CAN-12expression in the diseased tissue may suggest CAN-12 plays a defensiverole against disease progression, and agonists of CAN-12 polypeptidesmay be useful therapeutically in treating, preventing, and/orameliorating the disease. Also encompassed by the present invention arequantitative PCR probes corresponding to the polynucleotide sequenceprovided as SEQ ID NO:1 (FIGS. 1A-E).

The function of the protein may also be assessed through complementationassays in yeast. For example, in the case of the CAN-12, transformingyeast deficient in calpain activity, particularly m-calpain activity,and assessing their ability to grow would provide convincing evidencethe CAN-12 polypeptide has calpain activity, and possibly m-calpainactivity. Additional assay conditions and methods that may be used inassessing the function of the polynucleotides and polypeptides of thepresent invention are known in the art, some of which are disclosedelsewhere herein.

Alternatively, the biological function of the encoded polypeptide may bedetermined by disrupting a homologue of this polypeptide in Mice and/orrats and observing the resulting phenotype. Such knock-out experimentsare known in the art, some of which are disclosed elsewhere herein.

Moreover, the biological function of this polypeptide may be determinedby the application of antisense and/or sense methodology and theresulting generation of transgenic mice and/or rats. Expressing aparticular gene in either sense or antisense orientation in a transgenicmouse or rat could lead to respectively higher or lower expressionlevels of that particular gene. Altering the endogenous expressionlevels of a gene can lead to the observation of a particular phenotypethat can then be used to derive indications on the function of the gene.The gene can be either over-expressed or under expressed in every cellof the organism at all times using a strong ubiquitous promoter, or itcould be expressed in one or more discrete parts of the organism using awell characterized tissue-specific promoter (e.g., an esophagus, spinalcord, lymph node, thymus, or spleen specific promoter), or it can beexpressed at a specified time of development using an inducible and/or adevelopmentally regulated promoter.

In the case of CAN-12 transgenic mice or rats, if no phenotype isapparent in normal growth conditions, observing the organism underdiseased conditions (neural, immune, hematopoietic diseases ordisorders, cancers, etc.) may lead to understanding the function of thegene. Therefore, the application of antisense and/or sense methodologyto the creation of transgenic mice or rats to refine the biologicalfunction of the polypeptide is encompassed by the present invention.

In preferred embodiments, the following N-terminal CAN-12 deletionpolypeptides are encompassed by the present invention: M1-L581, S2-L581,L3-L581, W4-L581, P5-L581, P6-L581, F7-L581, R8-L581, C9-L581, R10-L581,W11-L581, K12-L581, L13-L581, A14-L581, P15-L581, R16-L581, Y17-L581,S18-L581, R19-L581, R20-L581, A21-L581, S22-L581, P23-L581, Q24-L581,Q25-L581, P26-L581, Q27-L581, Q28-L581, D29-L581, F30-L581, E31-L581,A32-L581, L33-L581, L34-L581, A35-L581, E36-L581, C37-L581, L38-L581,R39-L581, N40-L581, G41-L581, C42-L581, L43-L581, F44-L581, E45-L581,D46-L581, T47-L581, S48-L581, F49-L581, P50-L581, A51-L581, T52-L581,L53-L581, S54-L581, S55-L581, I56-L581, G57-L581, S58-L581, G59-L581,S60-L581, L61-L581, L62-L581, Q63-L581, K64-L581, L65-L581, P66-L581,P67-L581, R68-L581, L69-L581, Q70-L581, W71-L581, K72-L581, R73-L581,P74-L581, P75-L581, E76-L581, L77-L581, H78-L581, S79-L581, N80-L581,P81-L581, Q82-L581, F83-L581, Y84-L581, F85-L581, A86-L581, K87-L581,A88-L581, K89-L581, R90-L581, L91-L581, D92-L581, L93-L581, C94-L581,Q95-L581, G96-L581, I97-L581, V98-L581, G99-L581, D100-L581, C101-L581,W102-L581, F103-L581, L104-L581, A105-L581, A106-L581, L107-L581,Q108-L581, A109-L581, L110-L581, A111-L581, L112-L581, H113-L581,Q114-L581, D115-L581, I116-L581, L117-L581, S118-L581, R119-L581,V120-L581, V121-L581, P122-L581, L123-L581, N124-L581, Q125-L581,S126-L581, F127-L581, T128-L581, E129-L581, K130-L581, Y131-L581,A132-L581, G133-L581, I134-L581, F135-L581, R136-L581, F137-L581,W138-L581, F139-L581, W140-L581, H141-L581, Y142-L581, G143-L581,N144-L581, W145-L581, V146-L581, P147-L581, V148-L581, V149-L581,I150-L581, D151-L581, D152-L581, R153-L581, L154-L581, P155-L581,V156-L581, N157-L581, E158-L581, A159-L581, G160-L581, Q161-L581,L162-L581, V163-L581, F164-L581, V165-L581, S166-L581, S167-L581,T168-L581, Y169-L581, K170-L581, N171-L581, L172-L581, F173-L581,W174-L581, G175-L581, A176-L581, L177-L581, L178-L581, E179-L581,K180-L581, A181-L581, Y182-L581, A183-L581, K184-L581, L185-L581,S186-L581, G187-L581, S188-L581, Y189-L581, E190-L581, D191-L581,L192-L581, Q193-L581, S194-L581, G195-L581, Q196-L581, V197-L581,S198-L581, E199-L581, A200-L581, L201-L581, V202-L581, D203-L581,F204-L581, T205-L581, G206-L581, G207-L581, V208-L581, T209-L581,M210-L581, T211-L581, I212-L581, N213-L581, L214-L581, A215-L581,E216-L581, A217-L581, H218-L581, G219-L581, N220-L581, L221-L581,W222-L581, D223-L581, I224-L581, L225-L581, I226-L581, E227-L581,A228-L581, T229-L581, Y230-L581, N231-L581, R232-L581, T233-L581,L234-L581, I235-L581, G236-L581, C237-L581, Q238-L581, T239-L581,H240-L581, S241-L581, G242-L581, K243-L581, I244-L581, L245-L581,E246-L581, N247-L581, G248-L581, L249-L581, V250-L581, E251-L581,G252-L581, H253-L581, A254-L581, Y255-L581, T256-L581, L257-L581,T258-L581, G259-L581, I260-L581, R261-L581, K262-L581, V263-L581,T264-L581, C265-L581, K266-L581, H267-L581, R268-L581, P269-L581,E270-L581, Y271-L581, L272-L581, V273-L581, K274-L581, L275-L581,R276-L581, N277-L581, P278-L581, W279-L581, G280-L581, K281-L581,V282-L581, E283-L581, W284-L581, K285-L581, G286-L581, D287-L581,W288-L581, S289-L581, D290-L581, S291-L581, S292-L581, S293-L581,K294-L581, W295-L581, E296-L581, L297-L581, L298-L581, S299-L581,P300-L581, K301-L581, E302-L581, K303-L581, I304-L581, L305-L581,L306-L581, L307-L581, R308-L581, K309-L581, D310-L581, N311-L581,D312-L581-L581, G313-L581, E314-L581, F315-L581, W316-L581, M317-L581,T318-L581, L319-L581-L581, Q320-L581, D321-L581, F322-L581, K323-L581,T324-L581, H325-L581, F326-L581, V327-L581, L328-L581, L329-L581,V330-L581, I331-L581, C332-L581, K333-L581, L334-L581, T335-L581,P336-L581, G337-L581, L338-L581, L339-L581, S340-L581, Q341-L581,E342-L581, A343-L581, A344-L581, Q345-L581, K346-L581, W347-L581,T348-L581, Y349-L581, T350-L581, M351-L581, R352-L581, E353-L581,G354-L581, R355-L581, W356-L581, E357-L581, K358-L581, R359-L581,S360-L581, T361-L581, A362-L581, G363-L581, G364-L581, Q365-L581,R366-L581, Q367-L581, L368-L581, L369-L581, Q370-L581, D371-L581,T372-L581, F373-L581, W374-L581, K375-L581, N376-L581, P377-L581,Q378-L581, F379-L581, L380-L581, L381-L581, S382-L581, V383-L581,W384-L581, R385-L581, P386-L581, E387-L581, E388-L581, G389-L581,R390-L581, R391-L581, S392-L581, L393-L581, R394-L581, P395-L581,C396-L581, S397-L581, V398-L581, L399-L581, V400-L581, S401-L581,L402-L581, L403-L581, Q404-L581, K405-L581, P406-L581, R407-L581,H408-L581, R409-L581, C410-L581, R411-L581, K412-L581, R413-L581,K414-L581, P415-L581, L416-L581, L417-L581, A418-L581, I419-L581,G420-L581, F421-L581, Y422-L581, L423-L581, Y424-L581, R425-L581,M426-L581, N427-L581, K428-L581, M429-L581, T430-L581, W431-L581,S432-L581, S433-L581, L434-L581, G435-L581, S436-L581, R437-L581,Q438-L581, P439-L581, F440-L581, F441-L581, S442-L581, L443-L581,E444-L581, A445-L581, C446-L581, Q447-L581, G448-L581, I449-L581,L450-L581, A451-L581, L452-L581, L453-L581, D454-L581, L455-L581,N456-L581, A457-L581, S458-L581, G459-L581, T460-L581, M461-L581,S462-L581, I463-L581, Q464-L581, E465-L581, F466-L581, R467-L581,D468-L581, L469-L581, W470-L581, K471-L581, Q472-L581, L473-L581,K474-L581, L475-L581, S476-L581, Q477-L581, K478-L581, V479-L581,F480-L581, H481-L581, K482-L581, Q483-L581, D484-L581, R485-L581,G486-L581, S487-L581, G488-L581, Y489-L581, L490-L581, N491-L581,W492-L581, E493-L581, Q494-L581, L495-L581, H496-L581, A497-L581,A498-L581, M499-L581, R500-L581, E501-L581, A502-L581, G503-L581,R504-L581, H505-L581, R506-L581, K507-L581, S508-L581, W509-L581,S510-L581, C511-L581, G512-L581, H513-L581, T514-L581, R515-L581,A516-L581, G517-L581, C518-L581, T519-L581, L520-L581, I521-L581,R522-L581, Q523-L581, R524-L581, R525-L581, G526-L581, D527-L581,V528-L581, W529-L581, H530-L581, A531-L581, E532-L581, V533-L581,T534-L581, L535-L581, I536-L581, R537-L581, S538-L581, V539-L581,T540-L581, L541-L581, K542-L581, D543-L581, V544-L581, D545-L581,L546-L581, Q547-L581, S548-L581, T549-L581, P550-L581, T551-L581,F552-L581, F553-L581, M554-L581, I555-L581, V556-L581, P557-L581,V558-L581, I559-L581, L560-L581, A561-L581, N562-L581, I563-L581,D564-L581, G565-L581, G566-L581, V567-L581, A568-L581, H569-L581,S570-L581, T571-L581, S572-L581, Y573-L581, L574-L581, and/or I575-L581of SEQ ID NO:2. Polynucleotide sequences encoding these polypeptides arealso provided. The present invention also encompasses the use of theseN-terminal CAN-12 deletion polypeptides as immunogenic and/or antigenicepitopes as described elsewhere herein. The invention specificallyencompasses the N-terminal deletions from amino acid 1 to amino acid 421of SEQ ID NO:2.

In preferred embodiments, the following C-terminal CAN-12 deletionpolypeptides are encompassed by the present invention: M1-L581, M1-L580,M1-T579, M1-T578, M1-N577, M1-F576, M1-I575, M1-L574, M1-Y573, M1-S572,M1-T571, M1-S570, M1-H569, M1-A568, M1-V567, M1-G566, M1-G565, M1-D564,M1-I563, M1-N562, M1-A561, M1-L560, M1-I559, M1-V558, M1-P557, M1-V556,M1-I555, M1-M554, M1-F553, M1-F552, M1-T551, M1-P550, M1-T549, M1-S548,M1-Q547, M1-L546, M1-D545, M1-V544, M1-D543, M1-K542, M1-L541, M1-T540,M1-V539, M1-S538, M1-R537, M1-I536, M1-L535, M1-T534, M1-V533, M1-E532,M1-A531, M1-H530, M1-W529, M1-V528, M1-D527, M1-G526, M1-R525, M1-R524,M1-Q523, M1-R522, M1-I521, M1-L520, M1-T519, M1-C518, M1-G517, M1-A516,M1-R515, M1-T514, M1-H513, M1-G512, M1-C511, M1-S510, M1-W509, M1-S508,M1-K507, M1-R506, M1-H505, M1-R504, M1-G503, M1-A502, M1-E501, M1-R500,M1-M499, M1-A498, M1-A497, M1-H496, M1-L495, M1-Q494, M1-E493, M1-W492,M1-N491, M1-L490, M1-Y489, M1-G488, M1-S487, M1-G486, M1-R485, M1-D484,M1-Q483, M1-K482, M1-H481, M1-F480, M1-V479, M1-K478, M1-Q477, M1-S476,M1-L475, M1-K474, M1-L473, M1-Q472, M1-K471, M1-W470, M1-L469, M1-D468,M1-R467, M1-F466, M1-E465, M1-Q464, M1-I463, M1-S462, M1-M461, M1-T460,M1-G459, M1-S458, M1-A457, M1-N456, M1-L455, M1-D454, M1-L453, M1-L452,M1-A451, M1-L450, M1-I449, M1-G448, M1-Q447, M1-C446, M1-A445, M1-E444,M1-L443, M1-S442, M1-F441, M1-F440, M1-P439, M1-Q438, M1-R437, M1-S436,M1-G435, M1-L434, M1-S433, M1-S432, M1-W431, M1-T430, M1-M429, M1-K428,M1-N427, M1-M426, M1-R425, M1-Y424, M1-L423, M1-Y422, M1-F421, M1-G420,M1-I419, M1-A418, M1-L417, M1-L416, M1-P415, M1-K414, M1-R413, M1-K412,M1-R411, M1-C410, M1-R409, M1-H408, M1-R407, M1-P406, M1-K405, M1-Q404,M1-L403, M1-L402, M1-S401, M1-V400, M1-L399, M1-V398, M1-S397, M1-C396,M1-P395, M1-R394, M1-L393, M1-S392, M1-R391, M1-R390, M1-G389, M1-E388,M1-E387, M1-P386, M1-R385, M1-W384, M1-V383, M1-S382, M1-L381, M1-L380,M1-F379, M1-Q378, M1-P377, M1-N376, M1-K375, M1-W374, M1-F373, M1-T372,M1-D371, M1-Q370, M1-L369, M1-L368, M1-Q367, M1-R366, M1-Q365, M1-G364,M1-G363, M1-A362, M1-T361, M1-S360, M1-R359, M1-K358, M1-E357, M1-W356,M1-R355, M1-G354, M1-E353, M1-R352, M1-M351, M1-T350, M1-Y349, M1-T348,M1-W347, M1-K346, M1-Q345, M1-A344, M1-A343, M1-E342, M1-Q341, M1-S340,M1-L339, M1-L338, M1-G337, M1-P336, M1-T335, M1-L334, M1-K333, M1-C332,M1-1331, M1-V330, M1-L329, M1-L328, M1-V327, M1-F326, M1-H325, M1-T324,M1-K323, M1-F322, M1-D321, M1-Q320, M1-L319, M1-T318, M1-M317, M1-W316,M1-F315, M1-E314, M1-G313, M1-D312, M1-N311, M1-D310, M1-K309, M1-R308,M1-L307, M1-L306, M1-L305, M1-I304, M1-K303, M1-E302, M1-K301, M1-P300,M1-S299, M1-L298, M1-L297, M1-E296, M1-W295, M1-K294, M1-S293, M1-S292,M1-S291, M1-D290, M1-S289, M1-W288, M1-D287, M1-G286, M1-K285, M1-W284,M1-E283, M1-V282, M1-K281, M1-G280, M1-W279, M1-P278, M1-N277, M1-R276,M1-L275, M1-K274, M1-V273, M1-L272, M1-Y271, M1-E270, M1-P269, M1-R268,M1-H267, M1-K266, M1-C265, M1-T264, M1-V263, M1-K262, M1-R261, M1-I260,M1-G259, M1-T258, M1-L257, M1-T256, M1-Y255, M1-A254, M1-H253, M1-G252,M1-E251, M1-V250, M1-L249, M1-G248, M1-N247, M1-E246, M1-L245, M1-I244,M1-K243, M1-G242, M1-S241, M1-H240, M1-T239, M1-Q238, M1-C237, M1-G236,M1-I235, M1-L234, M1-T233, M1-R232, M1-N231, M1-Y230, M1-T229, M1-A228,M1-E227, M1-I226, M1-L225, M1-I224, M1-D223, M1-W222, M1-L221, M1-N220,M1-G219, M1-H218, M1-A217, M1-E216, M1-A215, M1-L214, M1-N213, M1-I212,M1-T211, M1-M210, M1-T209, M1-V208, M1-G207, M1-G206, M1-T205, M1-F204,M1-D203, M1-V202, M1-L201, M1-A200, M1-E199, M1-S198, M1-V197, M1-Q196,M1-195, M1-S194, M1-Q193, M1-L192, M1-D191, M1-E190, M1-Y189, M1-S188,M1-G187, M1-S186, M1-L185, M1-K184, M1-A183, M1-Y182, M1-A181, M1-K180,M1-E179, M1-L178, M1-L177, M1-A176, M1-G175, M1-W174, M1-F173, M1-L172,M1-N171, M1-K170, M1-Y169, M1-T168, M1-S167, M1-S166, M1-V165, M1-F164,M1-V163, M1-L162, M1-Q161, M1-G160, M1-A159, M1-E158, M1-N157, M1-V156,M1-P155, M1-L154, M1-R153, M1-D152, M1-D151, M1-I150, M1-V149, M1-V148,M1-P147, M1-V146, M1-W145, M1-N144, M1-G143, M1-Y142, M1-H141, M1-W140,M1-F139, M1-W138, M1-F137, M1-R136, M1-F135, M1-I134, M1-G133, M1-A132,M1-Y131, M1-K130, M1-E129, M1-T128, M1-F127, M1-S126, M1-Q125, M1-N124,M1-L123, M1-P122, M1-V121, M1-V120, M1-R119, M1-S118, M1-L117, M1-I116,M1-D115, M1-Q114, M1-H113, M1-L112, M1-A111, M1-L110, M1-A109, M1-Q108,M1-L107, M1-A106, M1-A105, M1-L104, M1-F103, M1-W102, M1-C101, M1-D100,M1-G99, M1-V98, M1-I97, M1-G96, M1-Q95, M1-C94, M1-L93, M1-D92, M1-L91,M1-R90, M1-K89, M1-A88, M1-K87, M1-A86, M1-F85, M1-Y84, M1-F83, M1-Q82,M1-P81, M1-N80, M1-S79, M1-H78, M1-L77, M1-E76, M1-P75, M1-P74, M1-R73,M1-K72, M1-W71, M1-Q70, M1-L69, M1-R68, M1-P67, M1-P66, M1-L65, M1-K64,M1-Q63, M1-L62, M1-L61, M1-S60, M1-G59, M1-S58, M1-G57, M1-I56, M1-S55,M1-S54, M1-L53, M1-T52, M1-A51, M1-P50, M1-F49, M1-S48, M1-T47, M1-D46,M1-E45, M1-F44, M1-L43, M1-C42, M1-G41, M1-N40, M1-R39, M1-L38, M1-C37,M1-E36, M1-A35, M1-L34, M1-L33, M1-A32, M1-E31, M1-F30, M1-D29, M1-Q28,M1-Q27, M1-P26, M1-Q25, M1-Q24, M1-P23, M1-S22, M1-A21, M1-R20, M1-R19,M1-S18, M1-Y17, M1-R16, M1-P15, M1-A14, M1-L13, M1-K12, M1-W11, M1-R10,M1-C9, M1-R8, and/or M1-F7 of SEQ ID NO:2. Polynucleotide sequencesencoding these polypeptides are also provided. The present inventionalso encompasses the use of these C-terminal CAN-12 deletionpolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein. The invention specifically encompasses the C-terminaldeletions from amino acid 428 to amino acid 7 of SEQ ID NO:2.

Alternatively, preferred polypeptides of the present invention maycomprise polypeptide sequences corresponding to, for example, internalregions of the CAN-12 polypeptide (e.g., any combination of both N— andC-terminal CAN-12 polypeptide deletions) of SEQ ID NO:2. For example,internal regions could be defined by the equation: amino acid NX toamino acid CX, wherein NX refers to any N-terminal deletion polypeptideamino acid of CAN-12 (SEQ ID NO:2), and where CX refers to anyC-terminal deletion polypeptide amino acid of CAN-12 (SEQ ID NO:2).Polynucleotides encoding these polypeptides are also provided. Thepresent invention also encompasses the use of these polypeptides as animmunogenic and/or antigenic epitope as described elsewhere herein.

The present invention also encompasses immunogenic and/or antigenicepitopes of the CAN-12 polypeptide.

The CAN-12 polypeptides of the present invention were determined tocomprise several phosphorylation sites based upon the Motif algorithm(Genetics Computer Group, Inc.). The phosphorylation of such sites mayregulate some biological activity of the CAN-12 polypeptide. Forexample, phosphorylation at specific sites may be involved in regulatingthe proteins ability to associate or bind to other molecules (e.g.,proteins, ligands, substrates, DNA, etc.). In the present case,phosphorylation may modulate the ability of the CAN-12 polypeptide toassociate with other polypeptides, particularly the serine proteasesubstrate for CAN-12, or its ability to modulate serine proteasefunction.

The CAN-12 polypeptide was predicted to comprise eleven PKCphosphorylation sites using the Motif algorithm (Genetics ComputerGroup, Inc.). In vivo, protein kinase C exhibits a preference for thephosphorylation of serine or threonine residues. The PKC phosphorylationsites have the following consensus pattern: [ST]-x-[RK], where S or Trepresents the site of phosphorylation and ‘x’ an intervening amino acidresidue. Additional information regarding PKC phosphorylation sites canbe found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem.161:177-184(1986), and Kishimoto A., Nishiyama K., Nakanishi H.,Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem.260:12492-12499(1985); which are hereby incorporated by referenceherein.

In preferred embodiments, the following PKC phosphorylation sitepolypeptides are encompassed by the present invention: LAPRYSRRASPQQ(SEQ ID NO:27), LNQSFTEKYAGIF (SEQ ID NO:28), VFVSSTYKNLFWG (SEQ IDNO:29), GCQTHSGKILENG (SEQ ID NO:30), GIRKVTCKHRPEY (SEQ ID NO:31),DWSDSSSKWELLS (SEQ ID NO:32), KWELLSPKEKILL (SEQ ID NO:33),QKWTYTMREGRWE (SEQ ID NO:34), EEGRRSLRPCSVL (SEQ ID NO:35),KQLKLSQKVFHKQ (SEQ ID NO:36), and/or LIRSVTLKDVDLQ (SEQ ID NO:37).Polynucleotides encoding these polypeptides are also provided. Thepresent invention also encompasses the use of the CAN-12 PKCphosphorylation site polypeptides as immunogenic and/or antigenicepitopes as described elsewhere herein.

The CAN-12 polypeptide has been shown to comprise four glycosylationsite according to the Motif algorithm (Genetics Computer Group, Inc.).As discussed more specifically herein, protein glycosylation is thoughtto serve a variety of functions including: augmentation of proteinfolding, inhibition of protein aggregation, regulation of intracellulartrafficking to organelles, increasing resistance to proteolysis,modulation of protein antigenicity, and mediation of intercellularadhesion.

In preferred embodiments, the following asparagine glycosylation sitepolypeptide is encompassed by the present invention: RVVPLNQSFTEKYA (SEQID NO:38), IEATYNRTLIGCQT (SEQ ID NO:39), ALLDLNASGTMSIQ (SEQ ID NO:40),and/or SYLWNTTLL (SEQ ID NO:41). Polynucleotides encoding thispolypeptide are also provided. The present invention also encompassesthe use of the CAN-12 asparagine glycosylation site polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

The CAN-12 polypeptide has been shown to comprise one amidation siteaccording to the Motif algorithm (Genetics Computer Group, Inc.). Theprecursor of hormones and other active peptides which are C-terminallyamidated is always directly followed by a glycine residue which providesthe amide group, and most often by at least two consecutive basicresidues (Arg or Lys) which generally function as an active peptideprecursor cleavage site. Although all amino acids can be amidated,neutral hydrophobic residues such as Val or Phe are good substrates,while charged residues such as Asp or Arg are much less reactive. Aconsensus pattern for amidation sites is the following: x-G-[RK]-[RK],wherein “X” represents the amidation site. Additional informationrelating to asparagine glycosylation may be found in reference to thefollowing publications, which are hereby incorporated by referenceherein: Kreil G., Meth. Enzymol. 106:218-223(1984); and Bradbury A. F.,Smyth D. G., Biosci. Rep. 7:907-916(1987).

In preferred embodiments, the following amidation site polypeptide isencompassed by the present invention: VWRPEEGRRSLRPC (SEQ ID NO:42).Polynucleotides encoding these polypeptides are also provided. Thepresent invention also encompasses the use of this CAN-12 amidation sitepolypeptide as an immunogenic and/or antigenic epitope as describedelsewhere herein.

The CAN-12 polypeptide has been shown to comprise one RGD cellattachment site domain according to the Motif algorithm (GeneticsComputer Group, Inc.). The sequence Arg-Gly-Asp, found in fibronectin,is crucial for its interaction with its cell surface receptor, anintegrin. What has been called the ‘RGD’ tripeptide is also found in thesequences of a number of other proteins, where it has been shown to playa role in cell adhesion. Non-limiting examples of these proteins are thefollowing: some forms of collagens, fibrinogen, vitronectin, vonWillebrand factor (VWF), snake disintegrins, and slime mold discoidins.The ‘RGD’ tripeptide is also found in other proteins where it may servethe same purpose. A consensus pattern for RGD cell attachment sites isthe following: R-G-D. Additional information relating to RGD cellattachment site domains may be found in reference to the followingpublications, which are hereby incorporated by reference herein:Ruoslahti E., Pierschbacher M. D., Cell 44:517-518(1986); and d'Souza S.E., Ginsberg M. H., Plow E. F., Trends Biochem. Sci. 16:246-250(1991).

In preferred embodiments, the following RGD cell attachment site domainpolypeptide is encompassed by the present invention: LIRQRRGDVWHAE (SEQID NO:43). Polynucleotides encoding these polypeptides are alsoprovided. The present invention also encompasses the use of this RGDcell attachment site domain polypeptide as an immunogenic and/orantigenic epitope as described elsewhere herein.

In confirmation of the CAN-12 polypeptide being a calpain, it has beenshown to comprise one EF-hand calcium-binding domain according to theMotif algorithm (Genetics Computer Group, Inc.). Many calcium-bindingproteins belong to the same evolutionary family and share a type ofcalcium-binding domain known as the EF-hand. This type of domainconsists of a twelve residue loop flanked on both side by a twelveresidue alpha-helical domain. In an EF-hand loop the calcium ion iscoordinated in a pentagonal bipyramidal configuration. The six residuesinvolved in the binding are in positions 1, 3, 5, 7, 9 and 12; theseresidues are denoted by X, Y, Z, —Y, —X and -Z. The invariant Glu or Aspat position 12 provides two oxygens for liganding Ca (bidentate ligand).Several representative proteins containing EF-hand regions are providedbelow: For each type of protein, the total number of EF-hand regionsknown or supposed to exist are provided in parenthesis: Aequorin andRenilla luciferin binding protein (LBP) (Ca=3); Alpha actinin (Ca=2);Calbindin (Ca=4); Calcineurin B subunit (protein phosphatase 2Bregulatory subunit) (Ca=4); Calcium-binding protein from Streptomyceserythraeus (Ca=3?); Calcium-binding protein from Schistosoma mansoni(Ca=2?); Calcium-binding proteins TCBP-23 and TCBP-25 from Tetrahymenathermophila (Ca=4?); Calcium-dependent protein kinases (CDPK) fromplants (Ca=4); Calcium vector protein from amphoxius (Ca=2); Calcyphosin(thyroid protein p24) (Ca=4?); Calmodulin (Ca=4, except in yeast whereCa=3); Calpain small and large chains (Ca=2); Calretinin (Ca=6);Calcyclin (prolactin receptor associated protein) (Ca=2); Caltractin(centrin) (Ca=2 or 4); Cell Division Control protein 31 (gene CDC3 1)from yeast (Ca=2?); Diacylglycerol kinase (EC 2.7.1.107) (DGK) (Ca=2);FAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) frommammals (Ca=1); Fimbrin (plastin) (Ca=2); Flagellar calcium-bindingprotein (lf8) from Trypanosoma cruzi (Ca=1 or 2); Guanylate cyclaseactivating protein (GCAP) (Ca=3); Inositol phospholipid-specificphospholipase C isozymes gamma-1 and delta-1 (Ca=2) [10]; Intestinalcalcium-binding protein (ICaBPs) (Ca=2); MIF related proteins 8 (MRP-8or CFAG) and 14 (MRP-14) (Ca=2); Myosin regulatory light chains (Ca=1);Oncomodulin (Ca=2); Osteonectin (basement membrane protein BM40) (SPARC)and proteins that contains an ‘osteonectin’ domain (QR1, matrixglycoprotein SC1) (Ca=1); Parvalbumins alpha and beta (Ca=2); Placentalcalcium-binding protein (18a2) (nerve growth factor induced protein 42a)(p9k) (Ca=2); Recoverins (visinin, hippocalcin, neurocalcin, S-modulin)(Ca=2 to 3); Reticulocalbin (Ca=4); S-100 protein, alpha and beta chains(Ca=2); Sarcoplasmic calcium-binding protein (SCPs) (Ca=2 to 3); Seaurchin proteins Spec 1 (Ca=4), Spec 2 (Ca=4?), Lps-1 (Ca=8);Serine/threonine protein phosphatase rdgc (EC 3.1.3.16) from Drosophila(Ca=2); Sorcin V19 from hamster (Ca=2); Spectrin alpha chain (Ca=2);Squidulin (optic lobe calcium-binding protein) from squid (Ca=4); andTroponins C; from skeletal muscle (Ca=4), from cardiac muscle (Ca=3),from arthropods and molluscs (Ca=2).

A consensus pattern for EF hand calcium binding domains is thefollowing: 1 2 3  4     5   6    7  8    9    10  12   13X   Y        Z       -Y     -X     -ZD-x-[DNS]-{ILVFYW}-[DENSTG]-[DNQGHRK]-{GP}-[LIVMC]-[DENQSTAGC]-x(2)-[DE]-[LIVMFYW],wherein X, Y, Z, —Y, —X, and -Z are as defined above, and wherein “x”represents any amino acid. Amino acid residues within the consensus atpositions 1 (X), 3 (Y) and 12 (-Z) are the most conserved. The 6thresidue in an EF-hand loop is in most cases a Gly.

Additional information relating to RGD cell attachment site domains maybe found in reference to the following publications, which are herebyincorporated by reference herein: Kawasaki H., Kretsinger R. H., ProteinProf. 2:305490(1995); Kretsinger R. H., Cold Spring Harbor Symp. Quant.Biol. 52:499-510(1987); Moncrief N. D., Kretsinger R. H., Goodman M., J.Mol. Evol. 30:522-562(1990); Nakayama S., Moncrief N. D., Kretsinger R.H., J. Mol. Evol. 34:416-448(1992); Heizmann C. W., Hunziker W., TrendsBiochem. Sci. 16:98-103(1991); Kligman D., Hilt D. C., Trends Biochem.Sci. 13:437-443(1988); Strynadka N. C. J., James M. N. G., Annu. Rev.Biochem. 58:951-98(1989); Haiech J., Sallantin J., Biochimie67:555-560(1985); Chauvaux S., Beguin P., Aubert J.-P., Bhat K. M., GowL. A., Wood T. M., Bairoch A., Biochem. J. 265:261-265(1990); BairochA., Cox J. A., FEBS Lett. 269:454-456(1990).

In preferred embodiments, the following EF-hand calcium binding domainpolypeptide is encompassed by the present invention:ILALLDLNASGTMSIQEFRDLWK (SEQ ID NO:44). Polynucleotides encoding thesepolypeptides are also provided. The present invention also encompassesthe use of this EF-hand calcium binding domain polypeptide as animmunogenic and/or antigenic epitope as described elsewhere herein.

In further confirmation of the CAN-12 polypeptide being a calpain, ithas been shown to comprise one eukaryotic thiol (cysteine) proteaseactive site domain according to the Motif algorithm (Genetics ComputerGroup, Inc.). Eukaryotic thiol proteases (EC 3.4.22.-) are a family ofproteolytic enzymes which contain an active site cysteine. Catalysisproceeds through a thioester intermediate and is facilitated by a nearbyhistidine side chain; an asparagine completes the essential catalytictriad. Non-limiting examples of proteases which are known to belong tothis family are provided below: Vertebrate lysosomal cathepsins B (EC3.4.22.1), H (EC 3.4.22.16), L (EC 3.4.22.15), and S (EC 3.4.22.27);Vertebrate lysosomal dipeptidyl peptidase I (EC 3.4.14.1) (also known ascathepsin C); Vertebrate calpains (EC 3.4.22.17) (Calpains areintracellular calcium-activated thiol protease that contain both aN-terminal catalytic domain and a C-terminal calcium-binding domain;Mammalian cathepsin K, which seems involved in osteoclastic boneresorption; Human cathepsin O; Bleomycin hydrolase (An enzyme thatcatalyzes the inactivation of the antitumor drug BLM (a glycopeptide);Plant enzymes: barley aleurain (EC 3.4.22.16), EP-B1/B4; kidney beanEP-C1, rice bean SH-EP; kiwi fruit actinidin (EC 3.4.22.14); papayalatex papain (EC 3.4.22.2), chymopapain (EC 3.4.22.6), caricain (EC3.4.22.30), and proteinase IV (EC 3.4.22.25); pea turgor-responsiveprotein 15A; pineapple stem bromelain (EC 3.4.22.32); rape COT44; riceoryzain alpha, beta, and gamma; tomato low-temperature induced,Arabidopsis thaliana A494, RD 1 9A and RD2 1 A; House-dust mitesallergens DerP 1 and EurM1; Cathepsin B-like proteinases from the wormsCaenorhabditis elegans (genes gcp-1, cpr-3, cpr4, cpr-5 and cpr-6),Schistosoma mansoni (antigen SM31) and Japonica (antigen SJ3 1),Haemonchus contortus (genes AC-1 and AC-2), and Ostertagia ostertagi(CP-1 and CP-3); Slime mold cysteine proteinases CP1 and CP2; Cruzipainfrom Trypanosoma cruzi and brucei; Throphozoite cysteine proteinase(TCP) from various Plasmodium species; Proteases from Leishmaniamexicana, Theileria annulata and Theileria parva; Baculovirusescathepsin-like enzyme (v-cath); Drosophila small optic lobes protein(gene sol), a neuronal protein that contains a calpain-like domain;Yeast thiol protease BLH1/YCP1I/LAP3; and Caenorhabditis eleganshypothetical protein C06G4.2, a calpain-like protein; Two bacterialpeptidases are also part of this family—Aminopeptidase C fromLactococcus lactis (gene pepC), and Thiol protease tpr fromPorphyromonas gingivalis.

A consensus pattern for eukaryotic thiol (cysteine) protease active sitedomains is the following: Q-x(3)-[GE]-x-C-[YW]-x(2)-[STAGC]-[STAGCV],wherein C is the active site residue, and “x” represents any amino acid.The residue in position 4 of the pattern is almost always cysteine; theonly exceptions are calpains (Leu), bleomycin hydrolase (Ser) and yeastYCP1 (Ser); while the residue in position 5 of the pattern is always Glyexcept in papaya protease IV where it is Glu.

An additional consensus pattern for eukaryotic thiol (cysteine) proteaseactive site domains is the following:[LIVMGSTAN]-x-H-[GSACE]-[LWM]-x-[LIVMAT](2)-G-x-[GSADNH], wherein H isthe active site residue, and “x” represents any amino acid.

An additional consensus pattern for eukaryotic thiol (cysteine) proteaseactive site domains is the following:[FYCH]-[WI]-[LIVT]-x-[KRQAG]-N-[ST]-W-x(3)-[FYW]-G-x(2)-G-[LFYW]-[LMFYG]-x-[LWMF], wherein N is the active site residue, and “x” representsany amino acid.

Additional information relating to for eukaryotic thiol (cysteine)protease active site domains may be found in reference to the followingpublications, which are hereby incorporated by reference herein: DufourE., Biochimie 70:1335-1342(1988); Kirschke H., Barrett A. J., RawlingsN. D., Protein Prof. 2:1587-1643(1995); Shi G.-P., Chapman H. A., BhairiS. M., Deleeuw C., Reddy V. Y., Weiss S. J., FEBS Lett.357:129-134(1995); Velasco G., Ferrando A. A., Puente X. S., Sanchez L.M., Lopez-Otin C., J. Biol. Chem. 269:27136-27142(1994); Chapot-ChartierM. P., Nardi M., Chopin M. C., Chopin A., Gripon J. C., Appl. Environ.Microbiol. 59:330-333(1993); Higgins D. G., McConnell D. J., Sharp P.M., Nature 340:604-604(1989); Rawlings N. D., Barrett A. J., Meth.Enzymol. 244:461-486(1994), and http://www.expasy.ch/cgi-bin/lists?peptidas.txt, which are hereby incorporated by reference intheir entirety herein.

In preferred embodiments, the following for eukaryotic thiol (cysteine)protease active site domain polypeptide is encompassed by the presentinvention: RLDLCQGIVGDCWFLAALQALA (SEQ ID NO:45). Polynucleotidesencoding these polypeptides are also provided. The present inventionalso encompasses the use of this for eukaryotic thiol (cysteine)protease active site domain polypeptide as an immunogenic and/orantigenic epitope as described elsewhere herein.

The present invention also provides a three-dimensional homology modelof the CAN-12 polypeptide (see FIG. 6) representing amino acids 12 to524 of CAN-12 (SEQ ID NO:2). As referenced herein, SEQ ID NO:2 comprisesadditional amino acids that are not part of the CAN-12 polypeptidesequence but have been added to the sequence for reference to the CAN-12splice variants referenced herein. The inclusion of the additional aminoacids also helps for a more complete homology model. A three-dimensionalhomology model can be constructed on the basis of the known structure ofa homologous protein (Greer et al, 1991, Lesk, et al, 1992, Cardozo, etal, 1995, Yuan, et al, 1995). The homology model of the CAN-12polypeptide, corresponding to amino acid residues 12 to 524 of SEQ IDNO:2, was based upon the homologous structure of CAN2, a m-calpainfamily member (Strobl et al, 2000; hCAN2; Genbank Accession No.gi|7546423; SEQ ID NO:11) and is defined by the set of structuralcoordinates set forth in Table IV herein.

The CAN-12 homology model of the present invention may provide one basisfor designing rational stimulators (agonists) and/or inhibitors(antagonists) of one or more of the biological functions of CAN-12, orof CAN-12 mutants having altered specificity (e.g., molecularly evolvedCAN-12 polypeptides, engineered site-specific CAN-12 mutants, CAN-12allelic variants, etc.).

Homology models are not only useful for designing rational agonistsand/or antagonists, but are also useful in predicting the function of aparticular polypeptide. The functional predictions from homology modelsare typically more accurate than the functional attributes derived fromtraditional polypeptide sequence homology alignments (e.g., CLUSTALW),particularly when the three dimensional structure of a relatedpolypeptide is known (e.g., m-calpain family member CAN2 protein;Genbank Accession No. gi|7546423; SEQ ID NO:11). The increasedprediction accuracy is based upon the fact that homology modelsapproximate the three-dimensional structure of a protein, while homologybased alignments only take into account the one dimension polypeptidesequence. Since the function of a particular polypeptide is determinednot only by its primary, secondary, and tertiary structure, functionalassignments derived solely upon homology alignments using the onedimensional protein sequence may be less reliable. A 3-dimensional modelcan be constructed on the basis of the known structure of a homologousprotein (Greer et al, 1991, Lesk, et al, 1992, Cardozo, et al, 1995,Yuan, et al, 1995).

Prior to developing a homology model, those of skill in the art wouldappreciate that a template of a known protein, or model protein, mustfirst be identified which will be used as a basis for constructing thehomology model for the protein of unknown structure (query template). Inthe case of the CAN-12 polypeptide of the present invention, the modelprotein template used in constructing the CAN-12 homology model was them-calpain family member CAN2 protein; Genbank Accession No. gi|7546423;SEQ ID NO:11).

Identifying a template can be accomplished using pairwise alignment ofprotein sequences using such programs as FASTA (Pearson, et al 1990) andBLAST (Altschul, et al, 1990). In cases where sequence similarity ishigh (greater than 30%), such pairwise comparison methods may beadequate for identifying an appropriate template. Likewise, multiplesequence alignments or profile-based methods can be used to align aquery sequence to an alignment of multiple (structurally andbiochemically) related proteins. When the sequence similarity is low,more advanced techniques may be used. Such techniques, include, forexample, protein fold recognition (protein threading; Hendlich, et al,1990), where the compatibility of a particular polypeptide sequence withthe 3-dimensional fold of a potential template protein is gauged on thebasis of a knowledge-based potential.

Following the initial sequence alignment, the second step would be tooptimally align the query template to the model template by manualmanipulation and/or by the incorporation of features specific to thepolypeptides (e.g., motifs, secondary structure predictions, and allowedconservations). Preferably, the incorporated features are found withinboth the model and query template.

The third step would be to identify structurally conserved regions thatcould be used to construct secondary core structure (Sali, et al, 1995).Loops could be added using knowledge-based techniques, and by performingforcefield calculations (Sali, et al, 1995).

The term “structure coordinates” refers to Cartesian coordinatesgenerated from the building of a homology model. In this invention, thehomology model of residues 12 to 524 of CAN-12 (SEQ ID NO:2) was derivedfrom generating a sequence alignment with m-calpain (Strobl et al, 2000;hCAN2; Genbank Accession No. gi|7546423; SEQ ID NO:11) using theCOMPOSER suite of software within SYBYL6.6 (Tripos Associates, St.Louis, Mo.) and then generating the backbone and side chainconformations. In the original crystal structure (pdb code 1dkv) as wellas the crystal structure reported elsewhere (Hosfield et al, 1999), theactive site of the enzyme comprising a cysteine, a histidine and anasparagine residue was not “formed”. The helix that contains the activesite C101 was altered by moving the helix down one pitch so that theactive site geometry could match that found in Papain (pdb code 1b4).This modified structure of human m-calpain was used as the template forconstruction of the homology model (illustrated in FIG. 6 herein).

The skilled artisan would appreciate that a set of structure coordinatesfor a protein represents a relative set of points that define a shape inthree dimensions. Thus, it is possible that an entirely different set ofcoordinates could define a similar or identical shape. Moreover, slightvariations in the individual coordinates, as emanate from the generationof similar homology models using different alignment templates (i.e.,other than the m-calpain (Strobl et al, 2000; hCAN2; Genbank AccessionNo. gi|7546423; SEQ ID NO:11), and/or using different methods ingenerating the homology model, will likely have minor effects on theoverall shape. Variations in coordinates may also be generated becauseof mathematical manipulations of the structure coordinates. For example,the structure coordinates set forth in Table IV could be manipulated byfractionalization of the structure coordinates; integer additions, orinteger subtractions to sets of the structure coordinates, inversion ofthe structure coordinates or any combination of the above.

Therefore, various computational analyses are necessary to determinewhether a template molecule or a portion thereof is sufficiently similarto all or part of a query template (e.g., CAN-12) in order to beconsidered the same. Such analyses may be carried out in currentsoftware applications, such as SYBYL version 6.6 or INSIGHTII (MolecularSimulations Inc., San Diego, Calif.) version 2000 and as described inthe accompanying User's Guides.

Using the superimposition tool in the program SYBYL, comparisons can bemade between different structures and different conformations of thesame structure. The procedure used in SYBYL to compare structures isdivided into four steps: 1) load the structures to be compared; 2)define the atom equivalencies in these structures; 3) perform a fittingoperation; and 4) analyze the results. Each structure is identified by aname. One structure is identified as the target (i.e., the fixedstructure); the second structure (i.e., moving structure) is identifiedas the source structure. The atom equivalency within SYBYL is defined byuser input. For the purpose of this invention, we will define equivalentatoms as protein backbone atoms (N, Cα, C and O) for all conservedresidues between the two structures being compared. We will alsoconsider only rigid fitting operations. When a rigid fitting method isused, the working structure is translated and rotated to obtain anoptimum fit with the target structure. The fitting operation uses analgorithm that computes the optimum translation and rotation to beapplied to the moving structure, such that the root mean squaredifference of the fit over the specified pairs of equivalent atoms is anabsolute minimum. This number, given in angstroms, is reported by theSYBYL program. For the purpose of the present invention, any homologymodel of a CAN-12 that has a root mean square deviation of conservedresidue backbone atoms (N, Cα, C, O) of less than 3.0 A whensuperimposed on the relevant backbone atoms described by structurecoordinates listed in Table IV are considered identical. Morepreferably, the root mean square deviation for the CAN-12 polypeptide isless than 2.0 Å.

The homology model of the present invention is useful for thestructure-based design of modulators of the CAN-12 biological function,as well as mutants with altered biological function and/or specificity.

In accordance with the structural coordinates provided in Table IV andthe three dimensional homology model of CAN-12, the CAN-12 polypeptidehas been shown to comprise a an active site region embodied by thefollowing amino acids: from about amino acid R90 to about amino acidC94, from about amino acid G99 to about amino acid L104, from aboutamino acid Q196 to about amino acid S198, from about amino acid M210 toabout amino acid I212, from about amino acid G236 to about amino acidH240, from about amino acid E251 to about amino acid Y255, from aboutamino acid N277 to about amino acid K281, from about amino acid V327 toabout amino acid V330 of SEQ ID NO:2 or SEQ ID NO:24 (FIGS. 1A-E). Inthis context, the term “about” may be construed to mean 1, 2, 3, 4, 5,6, 7, 8, 9 or 10 amino acids more in either the N— or C-terminaldirection of the above referenced amino acids.

Also more preferred are polypeptides comprising all or any part of theCAN-12 active site domain, or a mutant or homologue of said polypeptideor molecular complex. By mutant or homologue of the molecule is meant amolecule that has a root mean square deviation from the backbone atomsof said CAN-12 amino acids of not more than about 4.5 Angstroms, andpreferably not more than about 3.5 Angstroms.

In preferred embodiments, the following CAN-12 active site domainpolypeptide is encompassed by the present invention:RLDLCQGOVGDCWFLAALQALALHQDILSRVVPLNQSFTEKYAGIFRFWFWHYGNWVPVVIDDRLPVNEAGQLVFVSSTYKNLFWGALLEKAYAKLSGSYEDLQSGQVSEALVDFTGGVTMTINLAEAHGNLWDILIEATYNRTLIGCQTHSGKILENGLVEGHAYTLTGIRKVTCKHRPEYLVKLRNPWGKVEWKGDWSDSSSKWELLSPKEKILLLRKDNDGEFWMTLQDFKTHFVLLV (SEQ ID NO:46). Polynucleotides encoding thispolypeptide are also provided. The present invention also encompassesthe use of the CAN-12 active site domain polypeptide as an immunogenicand/or antigenic epitope as described elsewhere herein.

The present invention also encompasses polypeptides comprising at leasta portion of the CAN-12 active site domain (SEQ ID NO:46). Suchpolypeptides may correspond, for example, to the N— and/or C-terminaldeletions of the active site domain.

In preferred embodiments, the following N-terminal CAN-12 active sitedomain deletion polypeptides are encompassed by the present invention:R1-V241, L2-V241, D3-V241, L4-V241, C5-V241, Q6-V241, G7-V241, I8-V241,V9-V241, G 10-V241, D11-V241, C12-V241, W13-V241, F14-V241, L15-V241,A16-V241, A17-V241, L18-V241, Q19-V241, A20-V241, L21-V241, A22-V241,L23-V241, H24-V241, Q25-V241, D26-V241, I27-V241, L28-V241, S29-V241,R30-V241, V31-V241, V32-V241, P33-V241, L34-V241, N35-V241, Q36-V241,S37-V241, F38-V241, T39-V241, E40-V241, K41-V241, Y42-V241, A43-V241,G44-V241, I45-V241, F46-V241, R47-V241, F48-V241, W49-V241, F50-V241,W51-V241, H52-V241, Y53-V241, G54-V241, N55-V241, W56-V241, V57-V241,P58-V241, V59-V241, V60-V241, I61-V241, D62-V241, D63-V241, R64-V241,L65-V241, P66-V241, V67-V241, N68-V241, E69-V241, A70-V241, G71-V241,Q72-V241, L73-V241, V74-V241, F75-V241, V76-V241, S77-V241, S78-V241,T79-V241, Y80-V241, K81-V241, N82-V241, L83-V241, F84-V241, W85-V241,G86-V241, A87-V241, L88-V241, L89-V241, E90-V241, K91-V241, A92-V241,Y93-V241, A94-V241, K95-V241, L96-V241, S97-V241, G98-V241, S99-V241,Y100-V241, E101-V241, D102-V241, L103-V241, Q104-V241, S105-V241,G106-V241, Q107-V241, V108-V241, S109-V241, E110-V241, A111-V241,L112-V241, V113-V241, D114-V241, F115-V241, T116-V241-, G117-V241,G118-V241, V119-V241, T120-V241, M121-V241, T122-V241, I123-V241,N124-V241, L125-V241, A126-V241, E127-V241, A128-V241, H129-V241,G130-V241, N131-V241, L132-V241, W133-V241, D134-V241, I135-V241,L136-V241, I137-V241, E138-V241, A139-V241, T140-V241, Y141-V241,N142-V241, R143-V241, T144-V241, L145-V241, I146-V241, G147-V241,C148-V241, Q149-V241, T150-V241, H151-V241, S152-V241, G153-V241,K154-V241, I155-V241, L156-V241, E157-V241, N158-V241, G159-V241,L160-V241, V161-V241, E162-V241, G163-V241, H164-V241, A165-V241,Y166-V241, T167-V241, L168-V241, T169-V241, G170-V241, I171-V241,R172-V241, K173-V241, V174-V241, T175-V241, C176-V241, K177-V241,H178-V241, R179-V241, P180-V241, E181-V241, Y182-V241, L183-V241,V184-V241, K185-V241, L186-V241, R187-V241, N188-V241, P189-V241,W190-V241, G191-V241, K192-V241, V193-V241, E194-V241, W195-V241,K196-V241, G197-V241, D198-V241, W199-V241, S200-V241, D201-V241,S202-V241, S203-V241, S204-V241, K205-V241, W206-V241, E207-V241,L208-V241, L209-V241, S210-V241, P211-V241, K212-V241, E213-V241,K214-V241, I215-V241, L216-V241, L217-V241, L218-V241, R219-V241,K220-V241, D221-V241, N222-V241, D223-V241, G224-V241, E225-V241,F226-V241, W227-V241, M228-V241, T229-V241, L230-V241, Q231-V241,D232-V241, F233-V241, K234-V241, and/or T235-V241 of SEQ ID NO:46.Polynucleotide sequences encoding these polypeptides are also provided.The present invention also encompasses the use of these N-terminalCAN-12 active site domain deletion polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal CAN-12 active sitedomain deletion polypeptides are encompassed by the present invention:R1-V241, R1-L240, R1-L239, R1-V238, R1-F237, R1-H236, R1-T235, R1-K234,R1-F233, R1-D232, R1-Q231, R1-L230, R1-T229, R1-M228, R1-W227, R1-F226,R1-E225, R1-G224, R1-D223, R1-N222, R1-D221, R1-K220, R1-R219, R1-L218,R1-L217, R1-L216, R1-I215, R1-K214, R1-213, R1-K212, R1-P211, R1-S210,R1-L209, R1-L208, R1-E207, R1-W206, R1-K205, R1-S204, R1-S203, R1-S202,R1-D201, R1-S200, R1-W199, R1-D198, R1-G197, R1-K196, R1-W195, R1-E194,R1-V193, R1-K192, R1-G191, R1-W190, R1-P189, R1-N188, R1-R187, R1-L186,R1-K185, R1-V184, R1-L183, R1-Y182, R1-E181, R1-P180, R1-R179, R1-H178,R1-K177, R1-C176, R1-T175, R1-V174, R1-K173, R1-R172, R1-I171, R1-G170,R1-T169, R1-L168, R1-T167, R1-Y166, R1-A165, R1-H164, R1-G163, R1-E162,R1-V161, R1-L160, R1-G159, R1-N158, R1-E157, R1-L156, R1-I155, R1-K154,R1-G153, R1-S152, R1-H151, R1-T150, R1-Q149, R1-C148, R1-G147, R1-I146,R1-L145, R1-T144, R1-R143, R1-N142, R1-Y141, R1-T140, R1-A139, R1-E138,R1-I137, R1-L136, R1-I135, R1-D134, R1-W133, R1-L132, R1-N131, R1-G130,R1-H129, R1-A128, R1-E127, R1-A126, R1-L125, R1-N124, R1-I123, R1-T122,R1-M121, R1-T120, R1-V119, R1-G118, R1-G117, R1-T116, R1-F115, R1-D114,R1-V113, R1-L112, R1-A111, R1-E110, R1-S109, R1-V108, R1-Q107, R1-G106,R1-S105, R1-Q104, R1-L103, R1-D102, R1-E101, R1-Y100, R1-S99, R1-G98,R1-S97, R1-L96, R1-K95, R1-A94, R1-Y93, R1-A92, R1-K91, R1-E90, R1-L89,R1-L88, R1-A87, R1-G86, R1-W85, R1-F84, R1-L83, R1-N82, R1-K81, R1-Y80,R1-T79, R1-S78, R1-S77, R1-V76, R1-F75, R1-V74, R1-L73, R1-Q72, R1-G71,R1-A70, R1-E69, R1-N68, R1-V67, R1-P66, R1-L65, R1-R64, R1-D63, R1-D62,R1-I61, R1-V60, R1-V59, R1-P58, R1-V57, R1-W56, R1-N55, R1-G54, R1-Y53,R1-H52, R1-W51, R1-F50, R1-W49, R1-F48, R1-R47, R1-F46, R1-I45, R1-G44,R1-A43, R1-Y42, R1-K41, R1-E40, R1-T39, R1-F38, R1-S37, R1-Q36, R1-N35,R1-L34, R1-P33, R1-V32, R1-V31, R1-R30, R1-S29, R1-L28, R1-I27, R1-D26,R1-Q25, R1-H24, R1-L23, R1-A22, R1-L21, R1-A20, R1-Q19, R1-L18, R1-A17,R1-A16, R1-L15, R1-F14, R1-W13, R1-C12, R1-D11, R1-G10, R1-V9, R1-I8,and/or R1-G7 of SEQ ID NO:46. Polynucleotide sequences encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these C-terminal CAN-12 active site domain deletionpolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein.

Alternatively, such polypeptides may comprise polypeptide sequencescorresponding, for example, to internal regions of the CAN-12 activesite domain (e.g., any combination of both N— and C-terminal CAN-12active site domain deletions) of SEQ ID NO:46. For example, internalregions could be defined by the equation NX to CX, where NX refers toany N-terminal amino acid position of the CAN-12 active site domain (SEQID NO:46), and where CX refers to any C-terminal amino acid position ofthe CAN-12 active site domain (SEQ ID NO:46). Polynucleotides encodingthese polypeptides are also provided. The present invention alsoencompasses the use of these polypeptides as an immunogenic and/orantigenic epitope as described elsewhere herein.

In preferred embodiments, the following CAN-12 active site domain aminoacid substitutions are encompassed by the present invention: wherein R90is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q.S, T, V, W, or Y; wherein L91 is substituted with either an A, C, D, E,F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein D92 issubstituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein L93 is substituted with either an A, C, D, E, F,G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein C94 is substitutedwith either an A, D, E, F, G, H. I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein Q95 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, R, S, T, V, W, or Y; wherein G96 is substituted with eitheran A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein197 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q,R, S, T, V, W, or Y; wherein V98 is substituted with either an A, C, D,E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein G99 issubstituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein D100 is substituted with either an A, C, E, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein C101 is substitutedwith either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein W102 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, T, V, or Y; wherein F103 is substituted with eitheran A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinL104 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P,Q, R, S, T, V, W, or Y; wherein A105 is substituted with either a C, D,E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A106 issubstituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein L107 is substituted with either an A, C, D, E, F,G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein Q108 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, orY; wherein A109 is substituted with either a C, D, E, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein L110 is substituted with eitheran A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; whereinA111 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y; wherein L112 is substituted with either an A, C, D,E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein H113 issubstituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein Q114 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein D115 is substitutedwith either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein I116 is substituted with either an A, C, D, E, F, G, H, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein L117 is substituted with eitheran A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; whereinS118 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, T, V, W, or Y; wherein R119 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein V120 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, W, or Y; wherein V121 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein P122 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, orY; wherein L123 is substituted with either an A, C, D, E, F, G, H, I, K,M, N, P, Q, R, S, T, V, W, or Y; wherein N124 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, P, Q, P, S, T, V, W, or Y; whereinQ125 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, R, S, T, V, W, or Y; wherein S126 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein F127 issubstituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein T128 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein E129 is substitutedwith either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein K130 is substituted with either an A, C, D, E, F, G, H, I, L,M, N, P, Q, R, S, T, V, W, or Y; wherein Y131 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; whereinA132 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y; wherein G133 is substituted with either an A, C, D,E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I134 issubstituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein F135 is substituted with either an A, C, D, E, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R136 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, orY; wherein F137 is substituted with either an A, C, D, E, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein W138 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; whereinF139 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein W140 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein H141 issubstituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein Y142 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein G143 is substitutedwith either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein N144 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, P, Q, R, S, T, V, W, or Y; wherein W145 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; whereinV146 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, W, or Y; wherein P147 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein V148 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, W, or Y; wherein V149 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein I150 is substitutedwith either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein D151 is substituted with either an A, C, E, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein D152 is substituted with eitheran A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinR153 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, S, T, V, W, or Y; wherein L154 is substituted with either an A, C,D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein P155 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S,T, V, W, or Y; wherein V156 is substituted with either an A, C, D, E, F,O, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein N157 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; wherein E158 is substituted with either an A, C, D, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein A159 is substituted with eithera C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinG160 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein Q161 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein L162 issubstituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S,T, V, W, or Y; wherein V163 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein F164 is substitutedwith either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein V165 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, T, W, or Y; wherein S166 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; whereinS167 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, T, V, W, or Y; wherein T168 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein Y169 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, or W; wherein K170 is substituted with either an A, C, D, E, F,G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N171 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; wherein L172 is substituted with either an A, C, D, E, F, G, H, I, K,M, N, P, Q, R, S, T, V, W, or Y; wherein F173 is substituted with eitheran A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinW174 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, V, or Y; wherein G175 is substituted with either an A, C,D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A176 issubstituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein L177 is substituted with either an A, C, D, E, F,G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein L178 is substitutedwith either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY; wherein E179 is substituted with either an A, C, D, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein K180 is substituted with eitheran A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; whereinA181 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y; wherein Y182 is substituted with either an A, C, D,E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein A183 issubstituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein K184 is substituted with either an A, C, D, E, F,G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L185 is substitutedwith either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY; wherein S186 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, T, V, W, or Y; wherein G187 is substituted with eitheran A, C, D, E, F, H, I, K, L, M, N, P, Q, P, S, T, V, W, or Y; whereinS188 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, T, V, W, or Y; wherein Y189 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein E190 issubstituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein D191 is substituted with either an A, C, E, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L192 is substitutedwith either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY; wherein Q193 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, R, S, T, V, W, or Y; wherein S194 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; whereinG195 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein Q196 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein V197 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, W, or Y; wherein S198 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein E199 is substitutedwith either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein A200 is substituted with either a C, D, E, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein L201 is substituted with eitheran A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; whereinV202 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, W, or Y; wherein D203 is substituted with either an A, C,E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F204 issubstituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein T205 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein G206 is substitutedwith either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein G207 is substituted with either an A, C, D, E, F, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein V208 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; whereinT209 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, V, W, or Y; wherein M210 is substituted with either an A, C,D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; wherein T211 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, V, W, or Y; wherein 1212 is substituted with either an A, C, D, E, F,G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N213 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; wherein L214 is substituted with either an A, C, D, E, F, G, H, I, K,M, N, P, Q, R, S, T, V, W, or Y; wherein A215 is substituted with eithera C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinE216 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein A217 is substituted with either a C, D,E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein H218 issubstituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein G219 is substituted with either an A, C, D, E, F,H, I, K, L, M, N, P, Q. P, S, T, V, W, or Y; wherein N220 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; wherein L221 is substituted with either an A, C, D, E, F, G, H, I, K,M, N, P, Q, R, S, T, V, W, or Y; wherein W222 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; whereinD223 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein 1224 is substituted with either an A, C,D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L225 issubstituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S,T, V, W, or Y; wherein I226 is substituted with either an A, C, D, E, F,G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E227 is substitutedwith either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein A228 is substituted with either a C, D, E, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein T229 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; whereinY230 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, V, or W; wherein N231 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein R232 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S,T, V, W, or Y; wherein T233 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein L234 is substitutedwith either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY; wherein 1235 is substituted with either an A, C, D, E, F, G, H, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein G236 is substituted with eitheran A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinC237 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein Q238 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein T239 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, V, W, or Y; wherein H240 is substituted with either an A, C, D, E, F,G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S241 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, orY; wherein G242 is substituted with either an A, C, D, E, F, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein K243 is substituted with eitheran A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein1244 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein L245 is substituted with either an A, C,D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein E246 issubstituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein N247 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein G248 is substitutedwith either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein L249 is substituted with either an A, C, D, E, F, G, H, I, K,M, N, P, Q, R, S, T, V, W, or Y; wherein V250 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; whereinE251 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein G252 is substituted with either an A, C,D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein H253 issubstituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein A254 is substituted with either a C, D, E, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y255 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, orW; wherein T256 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, V, W, or Y; wherein L257 is substituted with eitheran A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; whereinT258 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, V, W, or Y; wherein G259 is substituted with either an A, C,D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I260 issubstituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein R261 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein K262 is substitutedwith either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, orY; wherein V263 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, T, W, or Y; wherein T264 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; whereinC265 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein K266 is substituted with either an A, C,D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein H267 issubstituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein R268 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein P269 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, orY; wherein E270 is substituted with either an A, C, D, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein Y271 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; whereinL272 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P,Q, R, S, T, V, W, or Y; wherein V273 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein K274 issubstituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S,T, V, W, or Y; wherein L275 is substituted with either an A, C, D, E, F,G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein R276 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, orY; wherein N277 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, P, Q, R, S, T, V, W, or Y; wherein P278 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; whereinW279 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, V, or Y; wherein G280 is substituted with either an A, C,D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K281 issubstituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S,T, V, W, or Y; wherein V282 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein E283 is substitutedwith either an A, C, D, F, G, H, I, K, L, M, N, P, Q. R, S, T, V, W, orY; wherein W284 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, T, V, or Y; wherein K285 is substituted with eitheran A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; whereinG286 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein D287 is substituted with either an A, C,E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W288 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, or Y; wherein S289 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein D290 is substitutedwith either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein S291 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, T, V, W, or Y; wherein S292 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; whereinS293 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, T, V, W, or Y; wherein K294 is substituted with either an A, C,D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W295 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, or Y; wherein E296 is substituted with either an A, C, D, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L297 is substitutedwith either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY; wherein L298 is substituted with either an A, C, D, E, F, G, H, I, K,M, N, P, Q, R, S, T, V, W, or Y; wherein S299 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; whereinP300 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,Q, R, S, T, V, W, or Y; wherein K301 is substituted with either an A, C,D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E302 issubstituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein K303 is substituted with either an A, C, D, E, F,G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I304 is substitutedwith either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein L305 is substituted with either an A, C, D, E, F, G, H, I, K,M, N, P, Q, R, S, T, V, W, or Y; wherein L306 is substituted with eitheran A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; whereinL307 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P,Q, R, S, T, V, W, or Y; wherein R308 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein K309 issubstituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S,T, V, W, or Y; wherein D310 is substituted with either an A, C, E, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N311 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; wherein D312 is substituted with either an A, C, E, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein G313 is substituted with eitheran A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinE314 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein F315 is substituted with either an A, C,D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W316 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, or Y; wherein M317 is substituted with either an A, C, D, E, F,G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; wherein T318 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, orY; wherein L319 is substituted with either an A, C, D, E, F, G, H, I, K,M, N, P, Q, R, S, T, V, W, or Y; wherein Q320 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; whereinD321 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein F322 is substituted with either an A, C,D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K323 issubstituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S,T, V, W, or Y; wherein T324 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein H325 is substitutedwith either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein F326 is substituted with either an A, C, D, E, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein V327 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; whereinL328 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P,Q, R, S, T, V, W, or Y; wherein L329 is substituted with either an A, C,D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; and/or whereinV330 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, W, or Y of SEQ ID NO:2 or SEQ ID NO:24, in addition toany combination thereof. The present invention also encompasses the useof these CAN-12 active site domain amino acid substituted polypeptidesas immunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following CAN-12 active site domainconservative amino acid substitutions are encompassed by the presentinvention: wherein R90 is substituted with either a K, or H; wherein L91is substituted with either an A, I, or V; wherein D92 is substitutedwith an E; wherein L93 is substituted with either an A, I, or V; whereinC94 is a C; wherein Q95 is substituted with a N; wherein G96 issubstituted with either an A, M, S, or T; wherein 197 is substitutedwith either an A, V, or L; wherein V98 is substituted with either an A,I, or L; wherein G99 is substituted with either an A, M, S, or T;wherein D100 is substituted with an E; wherein C101 is a C; wherein W102is either an F, or Y; wherein F103 is substituted with either a W, or Y;wherein L104 is substituted with either an A, I, or V; wherein A105 issubstituted with either a G, I, L, M, S, T, or V; wherein A106 issubstituted with either a G, I, L, M, S, T, or V; wherein L107 issubstituted with either an A, I, or V; wherein Q108 is substituted witha N; wherein A109 is substituted with either a G, I, L, M, S, T, or V;wherein L110 is substituted with either an A, I, or V; wherein A111 issubstituted with either a G, I, L, M, S, T, or V; wherein L112 issubstituted with either an A, I, or V; wherein H113 is substituted witheither a K, or R; wherein Q114 is substituted with a N; wherein D115 issubstituted with an E; wherein I116 is substituted with either an A, V,or L; wherein L117 is substituted with either an A, I, or V; whereinS118 is substituted with either an A, G, M, or T; wherein R119 issubstituted with either a K, or H; wherein V120 is substituted witheither an A, I, or L; wherein V121 is substituted with either an A, I,or L; wherein P122 is a P; wherein L123 is substituted with either an A,I, or V; wherein N124 is substituted with a Q; wherein Q125 issubstituted with a N; wherein S126 is substituted with either an A, G,M, or T; wherein F127 is substituted with either a W, or Y; wherein T128is substituted with either an A, G, M, or S; wherein E129 is substitutedwith a D; wherein K130 is substituted with either a R, or H; whereinY131 is either an F, or W; wherein A132 is substituted with either a G,I, L, M, S, T, or V; wherein G133 is substituted with either an A, M, S,or T; wherein I134 is substituted with either an A, V, or L; whereinF135 is substituted with either a W, or Y; wherein R136 is substitutedwith either a K, or H; wherein F137 is substituted with either a W, orY; wherein W138 is either an F, or Y; wherein F139 is substituted witheither a W, or Y; wherein W140 is either an F, or Y; wherein H141 issubstituted with either a K, or R; wherein Y142 is either an F, or W;wherein G143 is substituted with either an A, M, S, or T; wherein N144is substituted with a Q; wherein W145 is either an F, or Y; wherein V146is substituted with either an A, I, or L; wherein P147 is a P; whereinV148 is substituted with either an A, I, or L; wherein V149 issubstituted with either an A, I, or L; wherein I150 is substituted witheither an A, V, or L; wherein D151 is substituted with an E; whereinD152 is substituted with an E; wherein R153 is substituted with either aK, or H; wherein L154 is substituted with either an A, I, or V; whereinP155 is a P; wherein V156 is substituted with either an A, I, or L;wherein N157 is substituted with a Q; wherein E158 is substituted with aD; wherein A159 is substituted with either a G, I, L, M, S, T, or V;wherein G160 is substituted with either an A, M, S, or T; wherein Q161is substituted with a N; wherein L162 is substituted with either an A,I, or V; wherein V163 is substituted with either an A, I, or L; whereinF164 is substituted with either a W, or Y; wherein V165 is substitutedwith either an A, I, or L; wherein S166 is substituted with either an A,G, M, or T; wherein S167 is substituted with either an A, G, M, or T;wherein T168 is substituted with either an A, G, M, or S; wherein Y169is either an F, or W; wherein K170 is substituted with either a R, or H;wherein N171 is substituted with a Q; wherein L172 is substituted witheither an A, I, or V; wherein F173 is substituted with either a W, or Y;wherein W174 is either an F, or Y; wherein G175 is substituted witheither an A, M, S, or T; wherein A176 is substituted with either a G, I,L, M, S, T, or V; wherein L177 is substituted with either an A, I, or V;wherein L178 is substituted with either an A, I, or V; wherein E179 issubstituted with a D; wherein K180 is substituted with either a R, or H;wherein A181 is substituted with either a G, I, L, M, S, T, or V;wherein Y182 is either an F, or W; wherein A183 is substituted witheither a G, I, L, M, S, T, or V; wherein K184 is substituted with eithera R, or H; wherein L185 is substituted with either an A, I, or V;wherein S186 is substituted with either an A, G, M, or T; wherein G187is substituted with either an A, M, S, or T; wherein S188 is substitutedwith either an A, G, M, or T; wherein Y189 is either an F, or W; whereinE190 is substituted with a D; wherein D191 is substituted with an E;wherein L192 is substituted with either an A, I, or V; wherein Q193 issubstituted with a N; wherein S194 is substituted with either an A, G,M, or T; wherein G195 is substituted with either an A, M, S. or T;wherein Q196 is substituted with a N; wherein V197 is substituted witheither an A, I, or L; wherein S198 is substituted with either an A, G,M, or T; wherein E199 is substituted with a D; wherein A200 issubstituted with either a G, I, L, M, S, T, or V; wherein L201 issubstituted with either an A, I, or V; wherein V202 is substituted witheither an A, I, or L; wherein D203 is substituted with an E; whereinF204 is substituted with either a W, or Y; wherein T205 is substitutedwith either an A, G, M, or S; wherein G206 is substituted with either anA, M, S, or T; wherein G207 is substituted with either an A, M, S, or T;wherein V208 is substituted with either an A, I, or L; wherein T209 issubstituted with either an A, G, M, or S; wherein M210 is substitutedwith either an A, G, S, or T; wherein T211 is substituted with either anA, G, M, or S; wherein I212 is substituted with either an A, V, or L;wherein N213 is substituted with a Q; wherein L214 is substituted witheither an A, I, or V; wherein A215 is substituted with either a G, I, L,M, S, T, or V; wherein E216 is substituted with a D; wherein A217 issubstituted with either a G, I, L, M, S, T, or V; wherein H218 issubstituted with either a K, or R; wherein G219 is substituted witheither an A, M, S, or T; wherein N220 is substituted with a Q; whereinL221 is substituted with either an A, I, or V; wherein W222 is either anF, or Y; wherein D223 is substituted with an E; wherein I224 issubstituted with either an A, V, or L; wherein L225 is substituted witheither an A, I, or V; wherein I226 is substituted with either an A, V,or L; wherein E227 is substituted with a D; wherein A228 is substitutedwith either a G, I, L, M, S, T, or V; wherein T229 is substituted witheither an A, G, M, or S; wherein Y230 is either an F, or W; wherein N231is substituted with a Q; wherein R232 is substituted with either a K, orH; wherein T233 is substituted with either an A, G, M, or S; whereinL234 is substituted with either an A, I, or V; wherein I235 issubstituted with either an A, V, or L; wherein G236 is substituted witheither an A, M, S, or T; wherein C237 is a C; wherein Q238 issubstituted with a N; wherein T239 is substituted with either an A, G,M, or S; wherein H240 is substituted with either a K, or R; wherein S241is substituted with either an A, G, M, or T; wherein G242 is substitutedwith either an A, M, S, or T; wherein K243 is substituted with either aR, or H; wherein I244 is substituted with either an A, V, or L; whereinL245 is substituted with either an A, I, or V; wherein E246 issubstituted with a D; wherein N247 is substituted with a Q; wherein G248is substituted with either an A, M, S, or T; wherein L249 is substitutedwith either an A, I, or V; wherein V250 is substituted with either an A,I, or L; wherein E251 is substituted with a D; wherein G252 issubstituted with either an A, M, S, or T; wherein H253 is substitutedwith either a K, or R; wherein A254 is substituted with either a G, I,L, M, S, T, or V; wherein Y255 is either an F, or W; wherein T256 issubstituted with either an A, G, M, or S; wherein L257 is substitutedwith either an A, I, or V; wherein T258 is substituted with either an A,G, M, or S; wherein G259 is substituted with either an A, M, S, or T;wherein 1260 is substituted with either an A, V, or L; wherein R261 issubstituted with either a K, or H; wherein K262 is substituted witheither a R, or H; wherein V263 is substituted with either an A, I, or L;wherein T264 is substituted with either an A, G, M, or S; wherein C265is a C; wherein K266 is substituted with either a R, or H; wherein H267is substituted with either a K, or R; wherein R268 is substituted witheither a K, or H; wherein P269 is a P; wherein E270 is substituted witha D; wherein Y271 is either an F, or W; wherein L272 is substituted witheither an A, I, or V; wherein V273 is substituted with either an A, I,or L; wherein K274 is substituted with either a R, or H; wherein L275 issubstituted with either an A, I, or V; wherein R276 is substituted witheither a K, or H; wherein N277 is substituted with a Q; wherein P278 isa P; wherein W279 is either an F, or Y; wherein G280 is substituted witheither an A, M, S, or T; wherein K281 is substituted with either a R, orH; wherein V282 is substituted with either an A, I, or L; wherein E283is substituted with a D; wherein W284 is either an F, or Y; wherein K285is substituted with either a R, or H; wherein G286 is substituted witheither an A, M, S, or T; wherein D287 is substituted with an E; whereinW288 is either an F, or Y; wherein S289 is substituted with either an A,G, M, or T; wherein D290 is substituted with an E; wherein S291 issubstituted with either an A, G, M, or T; wherein S292 is substitutedwith either an A, G, M, or T; wherein S293 is substituted with either anA, G, M, or T; wherein K294 is substituted with either a R, or H;wherein W295 is either an F, or Y; wherein E296 is substituted with a D;wherein L297 is substituted with either an A, I, or V; wherein L298 issubstituted with either an A, I, or V; wherein S299 is substituted witheither an A, G, M, or T; wherein P300 is a P; wherein K301 issubstituted with either a R, or H; wherein E302 is substituted with a D;wherein K303 is substituted with either a R, or H; wherein I304 issubstituted with either an A, V, or L; wherein L305 is substituted witheither an A, I, or V; wherein L306 is substituted with either an A, I,or V; wherein L307 is substituted with either an A, I, or V; whereinR308 is substituted with either a K, or H; wherein K309 is substitutedwith either a R, or H; wherein D310 is substituted with an E; whereinN311 is substituted with a Q; wherein D312 is substituted with an E;wherein G313 is substituted with either an A, M, S, or T; wherein E314is substituted with a D; wherein F315 is substituted with either a W, orY; wherein W316 is either an F, or Y; wherein M317 is substituted witheither an A, G, S, or T; wherein T318 is substituted with either an A,G, M, or S; wherein L319 is substituted with either an A, I, or V;wherein Q320 is substituted with a N; wherein D321 is substituted withan E; wherein F322 is substituted with either a W, or Y; wherein K323 issubstituted with either a R, or H; wherein T324 is substituted witheither an A, G, M, or S; wherein H325 is substituted with either a K, orR; wherein F326 is substituted with either a W, or Y; wherein V327 issubstituted with either an A, I, or L; wherein L328 is substituted witheither an A, I, or V; wherein L329 is substituted with either an A, I,or V; and/or wherein V330 is substituted with either an A, I, or L ofSEQ ID NO:2 or SEQ ID NO:24 in addition to any combination thereof.Other suitable substitutions within the CAN-12 active site domain areencompassed by the present invention and are referenced elsewhereherein. The present invention also encompasses the use of these CAN-12active site domain conservative amino acid substituted polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

For purposes of the present invention, by “at least a portion of” ismeant all or any part of the CAN-12 active site domain defined by thestructure coordinates according to Table IV (e.g., fragments thereof).More preferred are molecules comprising all or any parts of the CAN-12active site domain, according to Table IV, or a mutant or homologue ofsaid molecule or molecular complex. By mutant or homologue of themolecule it is meant a molecule that has a root mean square deviationfrom the backbone atoms of said CAN-12 amino acids of not more than 4.5Angstroms, and preferably not more than 3.5 Angstroms.

The term “root mean square deviation” means the square root of thearithmetic mean of the squares of the deviations from the mean. It is aterm that expresses the deviation or variation from a trend or object.For the purposes of the present invention, the “root mean squaredeviation” defines the variation in the backbone of a protein from therelevant portion of the backbone of the AR portion of the complex asdefined by the structure coordinates described herein.

A preferred embodiment is a machine-readable data storage medium that iscapable of displaying a graphical three-dimensional representation of amolecule or molecular complex that is defined by the structurecoordinates of all of the amino acids in Table IV ± a root mean squaredeviation from the backbone atoms of those amino acids of not more than4.0 ANG, preferably 3.0 ANG.

The structure coordinates of a CAN-12 homology model, including portionsthereof, is stored in a machine-readable storage medium. Such data maybe used for a variety of purposes, such as drug discovery.

Accordingly, in one embodiment of this invention is provided amachine-readable data storage medium comprising a data storage materialencoded with the structure coordinates set forth in Table IV.

One embodiment utilizes System 10 as disclosed in WO 98/11134, thedisclosure of which is incorporated herein by reference in its entirety.Briefly, one version of these embodiments comprises a computercomprising a central processing unit (“CPU”), a working memory which maybe, e.g, RAM (random-access memory) or “core” memory, mass storagememory (such as one or more disk drives or CD-ROM drives), one or morecathode-ray tube (“CRT”) display terminals, one or more keyboards, oneor more input lines, and one or more output lines, all of which areinterconnected by a conventional bidirectional system bus.

Input hardware, coupled to the computer by input lines, may beimplemented in a variety of ways. Machine-readable data of thisinvention may be inputted via the use of a modem or modems connected bya telephone line or dedicated data line. Alternatively or additionally,the input hardware may comprise CD-ROM drives or disk drives. Inconjunction with a display terminal, keyboard may also be used as aninput device.

Output hardware, coupled to the computer by output lines, may similarlybe implemented by conventional devices. By way of example, outputhardware may include a CRT display terminal for displaying a graphicalrepresentation of a region or domain of the present invention using aprogram such as QUANTA as described herein. Output hardware might alsoinclude a printer, so that hard copy output may be produced, or a diskdrive, to store system output for later use.

In operation, the CPU coordinates the use of the various input andoutput devices, coordinates data accesses from mass storage, andaccesses to and from the working memory, and determines the sequence ofdata processing steps. A number of programs may be used to process themachine-readable data of this invention. Such programs are discussed inreference to the computational methods of drug discovery as describedherein. Specific references to components of the hardware system areincluded as appropriate throughout the following description of the datastorage medium.

For the purpose of the present invention, any magnetic data storagemedium which can be encoded with machine-readable data would besufficient for carrying out the storage requirements of the system. Themedium could be a conventional floppy diskette or hard disk, having asuitable substrate, which may be conventional, and a suitable coating,which may be conventional, on one or both sides, containing magneticdomains whose polarity or orientation could be altered magnetically, forexample. The medium may also have an opening for receiving the spindleof a disk drive or other data storage device.

The magnetic domains of the coating of a medium may be polarized ororiented so as to encode in a manner which may be conventional, machinereadable data such as that described herein, for execution by a systemsuch as the system described herein.

Another example of a suitable storage medium which could also be encodedwith such machine-readable data, or set of instructions, which could becarried out by a system such as the system described herein, could be anoptically-readable data storage medium. The medium could be aconventional compact disk read only memory (CD-ROM) or a rewritablemedium such as a magneto-optical disk which is optically readable andmagneto-optically writable. The medium preferably has a suitablesubstrate, which may be conventional, and a suitable coating, which maybe conventional, usually of one side of substrate.

In the case of a CD-ROM, as is well known, the coating is reflective andis impressed with a plurality of pits to encode the machine-readabledata. The arrangement of pits is read by reflecting laser light off thesurface of the coating. A protective coating, which preferably issubstantially transparent, is provided on top of the reflective coating.

In the case of a magneto-optical disk, as is well known, the coating hasno pits, but has a plurality of magnetic domains whose polarity ororientation can be changed magnetically when heated above a certaintemperature, as by a laser. The orientation of the domains can be readby measuring the polarization of laser light reflected from the coating.The arrangement of the domains encodes the data as described above.

Thus, in accordance with the present invention, data capable ofdisplaying the three dimensional structure of the CAN-12 homology model,or portions thereof and their structurally similar homologues is storedin a machine-readable storage medium, which is capable of displaying agraphical three-dimensional representation of the structure. Such datamay be used for a variety of purposes, such as drug discovery.

For the first time, the present invention permits the use ofstructure-based or rational drug design techniques to design, select,and synthesize chemical entities that are capable of modulating thebiological function of CAN-12.

Accordingly, the present invention is also directed to the design ofsmall molecules which imitates the structure of the CAN-12 active sitedomain (SEQ ID NO:46), or a portion thereof, in accordance with thestructure coordinates provided in Table IV. Alternatively, the presentinvention is directed to the design of small molecules which may bind toat least part of the CAN-12 active site domain (SEQ ID NO:25), or someportion thereof. For purposes of this invention, by CAN-12 active sitedomain, it is also meant to include mutants or homologues thereof. In apreferred embodiment, the mutants or homologues have at least 25%identity, more preferably 50% identity, more preferably 75% identity,and most preferably 90% identity to SEQ ID NO:46. In this context, theterm “small molecule” may be construed to mean any molecule describedknown in the art or described elsewhere herein, though may include, forexample, peptides, chemicals, carbohydrates, nucleic acids, PNAs, andany derivatives thereof.

The three-dimensional model structure of the CAN-12 will also providemethods for identifying modulators of biological function. Variousmethods or combination thereof can be used to identify these compounds.

For example, test compounds can be modeled that fit spatially into theactive site domain in CAN-12 embodied by the sequence from about R90 toabout amino acid C94, from about amino acid G99 to about amino acidL104, from about amino acid Q196 to about amino acid S198, from aboutamino acid M210 to about amino acid I212, from about amino acid G236 toabout amino acid H240, from about amino acid E251 to about amino acidY255, from about amino acid N277 to about amino acid K281, from aboutamino acid V327 to about amino acid V330, or some portion thereof, ofSEQ ID NO:2 or SEQ ID NO:24 (corresponding to SEQ ID NO:46), inaccordance with the structural coordinates of Table IV.

Structure coordinates of the active site domain in CAN-12 defined by theamino acids from about R90 to about amino acid C94, from about aminoacid G99 to about amino acid L104, from about amino acid Q196 to aboutamino acid S198, from about amino acid M210 to about amino acid I212,from about amino acid G236 to about amino acid H240, from about aminoacid E251 to about amino acid Y255, from about amino acid N277 to aboutamino acid K281, from about amino acid V327 to about amino acid V330 ofSEQ ID NO:2 or SEQ ID NO:24, can also be used to identify structural andchemical features. Identified structural or chemical features can thenbe employed to design or select compounds as potential CAN-12modulators. By structural and chemical features it is meant to include,but is not limited to, van der Waals interactions, hydrogen bondinginteractions, charge interaction, hydrophobic bonding interaction, anddipole interaction. Alternatively, or in conjunction with, thethree-dimensional structural model can be employed to design or selectcompounds as potential CAN-12 modulators. Compounds identified aspotential CAN-12 modulators can then be synthesized and screened in anassay characterized by binding of a test compound to the CAN-12, or incharacterizing the ability of CAN-12 to modulate a protease target inthe presence of a small molecule. Examples of assays useful in screeningof potential CAN-12 modulators include, but are not limited to,screening in silico, in vitro assays and high throughput assays.Finally, these methods may also involve modifying or replacing one ormore amino acids at amino acid positions, C101, H253, and/or N277 of SEQID NO:2 or SEQ ID NO:24 in accordance with the structure coordinates ofTable IV.

However, as will be understood by those of skill in the art upon thisdisclosure, other structure based design methods can be used. Variouscomputational structure based design methods have been disclosed in theart.

For example, a number computer modeling systems are available in whichthe sequence of the CAN-12 and the CAN-12 structure (i.e., atomiccoordinates of CAN-12 and/or the atomic coordinates of the active sitedomain as provided in Table IV) can be input. This computer system thengenerates the structural details of one or more these regions in which apotential CAN-12 modulator binds so that complementary structuraldetails of the potential modulators can be determined. Design in thesemodeling systems is generally based upon the compound being capable ofphysically and structurally associating with CAN-12. In addition, thecompound must be able to assume a conformation that allows it toassociate with CAN-12. Some modeling systems estimate the potentialinhibitory or binding effect of a potential CAN-12 modulator prior toactual synthesis and testing.

Methods for screening chemical entities or fragments for their abilityto associate with a given protein target are also well known. Oftenthese methods begin by visual inspection of the binding site on thecomputer screen. Selected fragments or chemical entities are thenpositioned in the active site domain of CAN-12. Docking is accomplishedusing software such as INSIGHTII, QUANTA and SYBYL, following by energyminimization and molecular dynamics with standard molecular mechanicforcefields such as CHARMM and AMBER. Examples of computer programswhich assist in the selection of chemical fragment or chemical entitiesuseful in the present invention include, but are not limited to, GRID(Goodford, 1985), AUTODOCK (Goodsell, 1990), and DOCK (Kuntz et al.1982).

Upon selection of preferred chemical entities or fragments, theirrelationship to each other and CAN-12 can be visualized and thenassembled into a single potential modulator. Programs useful inassembling the individual chemical entities include, but are not limitedto CAVEAT (Bartlett et al. 1989) and 3D Database systems (artin1992).

Alternatively, compounds may be designed de novo using either an emptyactive site or optionally including some portion of a known inhibitor.Methods of this type of design include, but are not limited to LUDI(Bohm 1992) and LeapFrog (Tripos Associates, St. Louis Mo.).

In addition, CAN-12 is overall well suited to modern methods includingcombinatorial chemistry.

Programs such as DOCK (Kuntz et al. 1982) can be used with the atomiccoordinates from the homology model to identify potential ligands fromdatabases or virtual databases which potentially bind CAN-12 active sitedomain, and which may therefore be suitable candidates for synthesis andtesting.

Additionally, the three-dimensional homology model of CAN-12 will aid inthe design of mutants with altered biological activity.

The following are encompassed by the present invention: amachine-readable data storage medium, comprising a data storage materialencoded with machine readable data, wherein the data is defined by thestructure coordinates of the model CAN-12 according to Table IV or ahomologue of said model, wherein said homologue comprises backbone atomsthat have a root mean square deviation from the backbone atoms of thecomplex of not more than 4.5 Å, preferably not more than 4.0 Å, mostpreferably not more than 3.5 Å, and even more preferably not more than3.0 Å; and a machine-readable data storage medium, wherein said moleculeis defined by the set of structure coordinates of the model for CAN-12according to Table IV, or a homologue of said molecule, said homologuehaving a root mean square deviation from the backbone atoms of saidamino acids of not more than 4.5 Å, preferably not more than 4.0 Å, mostpreferably not more than 3.5 Å, and even more preferably not more than3.0 Å; a model comprising all or any part of the model defined bystructure coordinates of CAN-12 according to Table IV, or a mutant orhomologue of said molecule or molecular complex.

In a further embodiment, the following are encompassed by the presentinvention: a method for identifying a mutant of CAN-12 with alteredbiological properties, function, or reactivity, the method comprisingany combination of steps of: use of the model or a homologue of saidmodel according to Table IV, for the design of protein mutants withaltered biological function or properties which exhibit any combinationof therapeutic effects provided elsewhere herein; and use of the modelor a homologue of said model, for the design of a protein with mutationsin the active site domain comprised of the amino acids from about R90 toabout amino acid C94, from about amino acid G99 to about amino acidL104, from about amino acid Q196 to about amino acid S198, from aboutamino acid M210 to about amino acid I212, from about amino acid G236 toabout amino acid H240, from about amino acid E251 to about amino acidY255, from about amino acid N277 to about amino acid K281, from aboutamino acid V327 to about amino acid V330 of SEQ ID NO:2 or SEQ ID NO:24according to Table IV with altered biological function or propertieswhich exhibit any combination of therapeutic effects provided elsewhereherein.

In further preferred embodiments, the following are encompassed by thepresent invention: a method for identifying modulators of CAN-12biological properties, function, or reactivity, the method comprisingany combination of steps of: modeling test compounds that overlayspatially into the active site domain defined by all or any portion ofresidues from about R90 to about amino acid C94, from about amino acidG99 to about amino acid L104, from about amino acid Q196 to about aminoacid S198, from about amino acid M210 to about amino acid I212, fromabout amino acid G236 to about amino acid H240, from about amino acidE251 to about amino acid Y255, from about amino acid N277 to about aminoacid K281, from about amino acid V327 to about amino acid V330 of SEQ IDNO:2 or SEQ ID NO:24 and of the three-dimensional structural modelaccording to Table IV, or using a homologue or portion thereof.

The present invention encompasses using the structure coordinates as setforth herein to identify structural and chemical features of the CAN-12polypeptide; employing identified structural or chemical features todesign or select compounds as potential CAN-12 modulators; employing thethree-dimensional structural model to design or select compounds aspotential CAN-12 modulators; synthesizing the potential CAN-12modulators; screening the potential CAN-12 modulators in an assaycharacterized by binding of a protein to the CAN-12; selecting thepotential CAN-12 modulator from a database; designing the CAN-12modulator de novo; and/or designing said CAN-12 modulator from a knownmodulator activity.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO: 1 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 4570 ofSEQ ID NO: 1, b is an integer between 15 to 4584, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ ID NO:1, and where b is greater than or equal to a+14.

In one embodiment, a CAN12 polypeptide comprises a portion of the aminosequence depicted in FIGS. 1A-E. In another embodiment, a CAN12polypeptide comprises at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 amino acids of the amino sequence depicted in FIGS.1A-E. In further embodiments, the CAN12 polypeptide does not consist ofthe sequence ALLEKAYAKL (SEQ ID NO: 141), and/or ALLEKAYAKLSGSYE. (SEQID NO: 142).

Features of the Polypeptide Encoded by Gene No:2

The polypeptide of this gene provided as SEQ ID NO:54 (FIGS. 8A-C),encoded by the polynucleotide sequence according to SEQ ID NO:53 (FIGS.8A-C), and/or encoded by the polynucleotide contained within thedeposited clone, CAN-12v1, has significant homology at the nucleotideand amino acid level to a number of calpains, which include, forexample, the human CAN10 protein (type II diabetes linked) (hCAN10;Genbank Accession No: gi|NP_(—)075574; SEQ ID NO:3); the human CAN5protein (hCAN5; Genbank Accession No: gi|NP_(—)004046; SEQ ID NO:4); thelarge catalytic subunit of the human CALPAIN 1 protein (also referred toas Calcium-Activated Neutral Proteinase, CANP, μ-TYPE) (hCAN1; GenbankAccession No: gi|12408656; SEQ ID NO:5); the large subunit of the humancalpain 3 protein (EC 3.4.22.17) (also referred to as CALPAIN L3,CALPAIN P94, Calcium-Activated Neutral Proteinase 3, CANP 3;muscle-specific calcium-activated neutral protease 3 large subunit)(hCAN3; Genbank Accession No: gi|4557405; SEQ ID NO:6); the largecatalytic subunit of the mouse CALPAIN 1 protein (also referred to asCalcium-Activated Neutral Proteinase) (CANP) μ-TYPE ) (mCALPAIN1;Genbank Accession No: gi|O88666; SEQ ID NO:7); the mouse CALPAIN LP82(mLP82; Genbank Accession No: gi|3661585; SEQ ID NO:8); the ratstomach-specific calcium-activated neutral protease large subunit, NCL2protein (rNCL2; Genbank Accession No: gi|NP_(—)006606; SEQ ID NO:9); thehuman CAN11 protein (hCAN11; Genbank Accession No: gi|NP008989; SEQ IDNO:10); the human CAN2 protein (hCAN2; Genbank Accession No: gi|4502563;SEQ ID NO:11); and the human CAN9 protein (hCAN9; Genbank Accession No:gi|5729758; SEQ ID NO:12). An alignment of the CAN-12v1 polypeptide withthese proteins is provided in FIGS. 2A-E. Based upon such strongconservation, the inventors have ascribed the CAN-12v1 polypeptide ashaving proteolytic activity, preferably calpain activity.

The CAN-12v1 polypeptide was determined to have 28.7% identity and 35.6%similarity with the human CAN10 protein (type II diabetes linked)(hCAN10; Genbank Accession No: gi|NP_(—)075574; SEQ ID NO:3); to have33.3% identity and 45.1% similarity with the human CAN5 protein (hCAN5;Genbank Accession No: gi|NP-004046; SEQ ID NO:4); to have 38.3% identityand 46.8% similarity with the large catalytic subunit of the humanCALPAIN 1 protein (also referred to as Calcium-Activated NeutralProteinase, CANP, μ-TYPE) (hCAN1; Genbank Accession No: gi|12408656; SEQID NO:5); to have 40.4% identity and 49.1% similarity with the largesubunit of the human calpain 3 protein (EC 3.4.22.17) (also referred toas CALPAIN L3, CALPAIN P94, Calcium-Activated Neutral Proteinase 3, CANP3; muscle-specific calcium-activated neutral protease 3 large subunit)(hCAN3; Genbank Accession No: gi|4557405; SEQ ID NO:6); to have 39.8%identity and 47.8% similarity with the large catalytic subunit of themouse CALPAIN 1 protein (also referred to as Calcium-Activated NeutralProteinase) (CANP) μ-TYPE ) (mCALPAIN1; Genbank Accession No: gi|O88666;SEQ ID NO:7); to have 40.6% identity and 48.8% similarity with the mouseCALPAIN LP82 (mLP82; Genbank Accession No: gi|3661585; SEQ ID NO:8); tohave 36.3% identity and 44.9% similarity with the rat stomach-specificcalcium-activated neutral protease large subunit, NCL2 protein (rNCL2;Genbank Accession No: gi|NP_(—)006606; SEQ ID NO:9); to have 38.8%identity and 47.4% similarity with the human CAN11 protein (hCAN11;Genbank Accession No: gi|NP_(—)008989; SEQ ID NO:10); to have 37.9%identity and 47.6% similarity with the human CAN2 protein (hCAN2;Genbank Accession No: gi|4502563; SEQ ID NO:11); and to have 40.7%identity and 49.8% similarity with the human CAN9 protein (hCAN9;Genbank Accession No: gi|5729758; SEQ ID NO:12).

The human CAN10 protein (type II diabetes linked) (hCAN10; GenbankAccession No: gi|NP_(—)075574; SEQ ID NO:3) is a human calpain gene thatencodes a large calpain subunit. CAN10 is an atypical calpain in that itlacks the calmodulin-like calcium-binding domain and instead has adivergent C-terminal domain. CAN10 is similar in organization tocalpains 5 and 6 and is associated with type 2 or non-insulin-dependentdiabetes mellitus (NIDDM) and located within the NIDDM1 chromosomalregion (Nat. Genet. 26 (2), 163-175 (2000)).

The large subunit of the human calpain 3 protein (EC 3.4.22.17) (alsoreferred to as CALPAIN L3, CALPAIN P94, Calcium-Activated NeutralProteinase 3, CANP 3; muscle-specific calcium-activated neutral protease3 large subunit) (hCAN3; Genbank Accession No: gi|4557405; SEQ ID NO:6)is a muscle-specific member of the calpain large subunit family. Loss ofCAPN3 function has been associated with limb-girdle muscular dystrophiestype 2A (Cell 81 (1), 27-40 (1995)).

The human CAN9 protein (hCAN9; Genbank Accession No: gi|5729758; SEQ IDNO:12) is a calpain that is expressed predominantly in stomach and smallintestine and is thought to have specialized functions in the digestivetract, and be associated with gastric cancer. (Biol. Chem. 379 (2),175-183 (1998); and Jpn. J. Cancer Res. 91 (5), 459-463 (2000)).

As described above, the CAN-12v1 polypeptide was found to havesignificant sequence homology with calpains, particularly members of them-calpain family. A conserved peptide signature ofQ×3(G,E)×C(Y,W)×2(S,T,A,G,C)(S,T,A,G,C,V) Q×{3}(G)×C(W)×{2}(A)(A)(referred to as a thiol (cysteine) protease active site domain) commonto most calpain family members is found in the protein sequence ofCAN-12v1 from amino acid 90 to amino acid 111 of SEQ ID NO:54 (FIGS.1A-C). Protein threading and molecular modeling of CAN-12v1 suggeststhat CAN-12v1 has a structural fold similar to representativem-calpains. Moreover, the structural and threading alignments of thepresent invention suggest that-amino acids 101 (“C”), 254 (“H”), and 278(“N”) of SEQ ID NO:54 (FIGS. 8A-C) may represent the catalytic aminoacids within the active site domain. Thus, based upon the sequence andstructural homology to known calpains, particularly the presence of thethiol cysteine protease active site domain, the novel CAN-12v1 isbelieved to represent a novel human calpain.

In confirmation of the strong homology to known calpains, the CAN-12v1polypeptide was determined to have several conserved catalytic aminoacids at amino acid C101, H254, and N278 of SEQ ID NO:54 (FIGS. 8A-C).As discussed more particularly herein, calpains are a group ofstructurally diverse, high molecular weight (400 to 500 amino acids)proteins that have a catalytic cysteine amino acid and one or morecalcium binding domains. Despite the structural heterogeneity, calpainsshare some well defined structural-functional characteristics,particularly in their active site domains.

In preferred embodiments, the CAN-12v1 polypeptide of the presentinvention is directed to a polypeptide having structural similarity tocalpains.

Based upon the strong homology to members of the calpain family, theCAN-12v1 polypeptide is expected to share at least some biologicalactivity with calpains, preferably with m-calpain family members, andmore preferable to the large subunits of m-calpain family members, inaddition to other calpains and calpain subunits referenced herein and/orotherwise known in the art.

Expression profiling designed to measure the steady state mRNA levelsencoding the CAN-12 polypeptide showed predominately high expressionlevels in spinal cord tissue; significantly high expression in lymphnode and thymus, and to a lesser extent, in spleen tissue (See FIG. 4).

Expanded analysis of CAN-12 expression levels by TaqMan™ quantitativePCR (see FIG. 6) confirmed that the CAN-12 polypeptide is expressed inthe lymph gland. However, the TaqMan™ quantitative PCR determined thatthe CAN-12 polypeptide is primarily expressed in the eosophagus. Infact, with the exception of the lymph gland, the steady state mRNA levelof CAN-12 was approximately 2700 times higher in the esophagus than inall other tissues tested. These data suggest modulators of the CAN-12polynucleotides and polypeptides may be useful for the treatment,detection, and/or amelioration of the following, non-limiting diseasesand disorders associated with the eosphagus: dysphagia, cricoharyngealincoordination, esophageal carcinoma, esophageal webs, achalasia,symptomatic diffuse esophageal spasm, gastroesophageal reflux, and/orcorrosive esophagitis.

The polynucleotides encoding the CAN-12 polypeptide of the presentinvention were used to determine the chromosomal localization of thecalpain12 gene. Polynuclelotides corresponding to CAN-12 (SEQ ID NO:1)were shown to localize to chromosome 2, specifically 2p16-p21. Thecomparison of the chromosomal location of the calpain12 gene with thelocation of chromosomal regions which have been shown to be associatedwith specific diseases or conditions, e.g. by linkage analysis, can beindicative of diseases in which calpain12 may play a role.Interestingly, a whole-genome linkage scan in multiple sclerosisfamilies (Ebers et al. A full genome search in multiple sclerosis.Nature Genet. 13: 472-476, 1996.) identified 5 susceptibility loci onchromosomes 2, 3, 5, 11, and X. In particular, an association wasidentified with marker D2S119 on chromosome 2 and MS, The localizationof the D2 S119 marker was further delimeated to 2p16-p21 based on aradiation hybrid linkage map retrieved from an online query at NCBI website: http:/www.ncbi.nlm.nih.gov/genome/sts/. Since the map of calpain12 and the susceptibility marker D2S119 overlaps, it is reasonable topostulate that calpain 12 may contribute to MS. Furthermore, thetranscription profile of calpain12 indicated a prominent expression inspinal cord, and implication of calpains in MS has been suggested(Shields D C et al. A putative mechanism of demyelination in multiplesclerosis by a proteolytic enzyme, calpain. Proc Natl Acad Sci U S A.96:11486-91.1999).

The CAN-12v1 polynucleotides and polypeptides of the present invention,including agonists and/or fragments thereof, have uses that includemodulating cellular adhesion events, cellular proliferation, andinflammation, in various cells, tissues, and organisms, and particularlyin mammalian spinal cord tissue, lymph node, thymus, and spleen tissue,preferably human tissue. CAN-12v1 polynucleotides and polypeptides ofthe present invention, including agonists and/or fragments thereof, maybe useful in diagnosing, treating, prognosing, and/or preventing neural,immune, hematopoietic, and/or proliferative diseases or disorders.

The strong homology to human calpains, particularly m-calpains, combinedwith the predominate localized expression in esophagus tissue suggeststhe CAN-12v1 polynucleotides and polypeptides may be useful in treating,diagnosing, prognosing, and/or preventing gastrointestinal diseases,particularly esophageal diseases and/or disorders which include thefollowing non-limiting examples: aberrant transport of food bolus fromthe mouth to the stomach, aberrant prevention of retrograde flow ofgastrointestinal contents, aberrant esophageal peristaltic contractions,pyrosis, painful swallowing, reflux esophagitis, esophageal motilitydisorders, esophageal spasms, diffuse esophageal spasm, atypical chestpain, regurgitation, oropharyngeal paralysis, nasal regurgitation,dysphagia, cricopharyngeal bar, globus pharyngeus, achalasia, motordisorders of the esophageal smooth muscle, scleroderma esophagus,gastroesophageal reflux disease (GERD), esophagitis, Barrett'sesophagus, viral esophagitis, Herpes simplex virus mediated viralesophagitis, Varicella-zoster virus mediated viral esophagitis,Cytomegalovirus mediated viral esophagitis, bacterial esophagitis,Lactobacillus mediated bacterial esophagitis, Candida mediatedesophagitis, radiation esophagitis, corrosive esophagitis, pill-inducedesophagitis, esophagitis associated with mucocutaneous and systemicdiseases, diverticula, lower esophageal mucosal ring, lower esophagealmuscular ring, hiatal hernia, paraesophageal hernia, esophageal rupture,and/or Mallory-Weiss Syndrome.

Although calpains are typically associated primarily withneurogenerative conditions, their association in gastrointenstinaltissues has precedence. For example, the human CAN9 protein (hCAN9;Genbank Accession No: gi|5729758; SEQ ID NO:12) is predominatelyexpressed in the stomach and small intestine and is thought to beassociated with gastric cancers.

The strong homology to human calpains, particularly m-calpains, combinedwith the localized expression in spinal cord tissue suggests theCAN-12v1 polynucleotides and polypeptides may be useful in treating,diagnosing, prognosing, and/or preventing neural diseases,neurodegenerative disease states, behavioral disorders, or inflammatoryconditions. Representative uses are described in the “NeurologicalDiseases”, “Regeneration” and “Hyperproliferative Disorders” sectionsbelow, in the Examples, and elsewhere herein. Briefly, the uses include,but are not limited to the detection, treatment, and/or prevention ofAlzheimer's Disease, Parkinson's Disease, Huntington's Disease, TouretteSyndrome, meningitis, encephalitis, demyelinating diseases, peripheralneuropathies, neoplasia, trauma, congenital malformations, spinal cordinjuries, ischemia and infarction, aneurysms, hemorrhages,schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder,depression, panic disorder, learning disabilities, ALS, psychoses,autism, and altered behaviors, including disorders in feeding, sleeppatterns, balance, and perception. In addition, elevated expression ofthis gene product in regions of the brain indicates it plays a role innormal neural function. Potentially, this gene product is involved insynapse formation, neurotransmission, learning, cognition, homeostasis,or neuronal differentiation or survival. Furthermore, the protein mayalso be used to determine biological activity, to raise antibodies, astissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

Alternatively, the strong homology to human calpains, particularlym-calpains, combined with the localized expression in lymph node,thymus, and spleen tissue suggests the CAN-12v1 polynucleotides andpolypeptides may be useful in treating, diagnosing, prognosing,ameliorating, and/or preventing immune diseases and/or disorders.Representative uses are described in the “Immune Activity” and“Infectious Disease” sections below, and elsewhere herein. Briefly, thestrong expression in immune tissue indicates a role in regulating theproliferation; survival; differentiation; and/or activation ofhematopoietic cell lineages, including blood stem cells. The CAN-12v1polypeptide may also be useful as a preventative agent for immunologicaldisorders including arthritis, asthma, immunodeficiency diseases such asAIDS, leukemia, rheumatoid arthritis, granulomatous disease,inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia,psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity;immune reactions to transplanted organs and tissues, such ashost-versus-graft and graft-versus-host diseases, or autoimmunitydisorders, such as autoimmune infertility, lense tissue injury,demyelination, systemic lupus erythematosis, drug induced hemolyticanemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.Moreover, the protein may represent a secreted factor that influencesthe differentiation or behavior of other blood cells, or that recruitshematopoietic cells to sites of injury. Thus, this gene product may beuseful in the expansion of stem cells and committed progenitors ofvarious blood lineages, and in the differentiation and/or proliferationof various cell types.

Moreover, the protein would be useful in the detection, treatment,and/or prevention of a variety of vascular disorders and conditions,which include, but are not limited to miscrovascular disease, vascularleak syndrome, aneurysm, stroke, embolism, thrombosis, coronary arterydisease, arteriosclerosis, and/or atherosclerosis. Furthermore, theprotein may also be used to determine biological activity, raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

In addition, antagonists of the CAN-12v1 polynucleotides andpolypeptides may have uses that include diagnosing, treating,prognosing, and/or preventing diseases or disorders related to hypercalpain activity, which may include immune and/or proliferative diseasesor disorders, particularly thrombosis, embolism, and other blooddisorders. Therapeutic and/or pharmaceutical compositions comprising theCAN-12v1 polypeptides may be formulated to comprise heparin.

In addition, antagonists of the CAN-12v1 polynucleotides andpolypeptides may have uses that include diagnosing, treating,ameliorating, prognosing, and/or preventing diseases or disordersrelated to hyper calpain activity, which may include neuronalexcitotoxicity, ischemic stroke, hemoragic stroke, hypoxic stress,trauma, cell destruction, spinal cord injury following trauma,degeneration of vulnerable hippocampal neurons after ischemia,reovirus-induced apoptosis, viral-induced induced myocarditis, acute andchronic inflammation, cataract formation, multiple sclerosis,demylenating disorders, acoustic trauma, hearing loss caused by noise,neuronal damage, cardiac ischemic damage, and/or hepatocyte necrosisduring and following anoxia

CAN-12v1 polynucleotides and polypeptides, including fragments and/orantagonsists thereof, may have uses which include modulatingdevelopment, differentiation, cellular transformation in response tocell signaling, cell-cell and/or cell-extracellular matrix interactions,clustering of the integrin receptor aIIb3, modulating in long termpotentiation (memory), modulating neurite outgrowth, modulating corticallamination activation of protein kinases and phosphatases, remodelingand disassembling the cytoskeleton, cell cycle modulation, in addition,to ameliorating, preventing, and/or treating limb-girdle musculardystrophy (LGMD), insulin resistance in diabetics, Alzheimer's disease,Multiple sclerosis, Huntington's disease, Parkinson's disease andamyotrophy.

Moreover, CAN-12v1 polynucleotides and polypeptides, including fragmentsand agonists thereof, may have uses which include treating, diagnosing,prognosing, and/or preventing hyperproliferative disorders, particularlyof the neural and immune systems. Such disorders may include, forexample, cancers, and metastatic conditions.

CAN-12v1 polynucleotides and polypeptides, including fragments and/orantagonsists thereof, may have uses which include identification ofmodulators of CAN-12v1 function including antibodies (for detection orneutralization), naturally-occurring modulators and small moleculemodulators. Antibodies to domains (including CAN-12v1 epitopes providedherein) of the CAN-12v1 protein could be used as diagnostic agents ofinflammatory conditions in patients, are useful in monitoring theactivation and presence of cognate proteases, and can be used as abiomarker for the protease involvement in disease states and in theevaluation of inhibitors of the cognate protease in vivo.

CAN-12v1 polypeptides and polynucleotides are useful for diagnosingdiseases related to over or under expression of CAN-12v1 proteins byidentifying mutations in the CAN-12v1 gene using CAN-12v1 probes, ordetermining CAN-12v1 protein or mRNA expression levels. CAN-12v1polypeptides are also useful for screening for compounds, which affectactivity of the protein. Diseases that can be treated with CAN-12v1include, the following, non-limiting examples: neuro-regeneration,neuropathic pain, obesity, anorexia, HIV infections, cancers, bulimia,asthma, Parkinson's disease, acute heart failure, hypotension,hypertension, osteoporosis, angina pectoris, myocardial infarction,psychotic, immune, metabolic, cardiovascular, and neurologicaldisorders.

The predominate expression in neural tissues, combined with thesignificant expression in a number of other tissues, suggests theCAN-12v1 polynucleotide and polypeptide of the present invention may beinvolved in modulating nerve invasion, innervation, nerve maintenance,and potentially myeline sheath maintenance and integrity.

The CAN-12v1 polynucleotides and polypeptides, including fragments andantagonists thereof, may have uses which include detecting, diagnosing,treating, ameliorating, and/or preventing diseases and disorders of theneural system, particularly Alzheimer's disease, either directly orindirectly, in addition to other neural disorders known in the art orprovided in the “Neurological Diseases” section herein, such asmodulating nerve invasion, innervation, nerve maintenance, potentiallymyelin sheath maintenance and integrity, encephalomyelitis, autoimmuneencephalomyelitis, human T cell leukemia virus type I(HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP),and neuro-inflammatory diseases.

Molecular genetic manipulation of the structure of the active sitedomain, particularly the predicted catalytic amino acids, and of otherfunctional domains in the calpain family (e.g., active site domainbinding pocket) enables the production of calpains with tailor-madeactivities. Thus, the CAN-12v1 polypeptides, and fragments thereof, aswell as any homologous product resulting from genetic manipulation ofthe structure, are useful for NMR-based design of modulators of CAN-12v1biological activity, and calpains, in general.

CAN-12v1 polypeptides and polynucleotides have additional uses whichinclude diagnosing diseases related to the over and/or under expressionof CAN-12v1 by identifying mutations in the CAN-12v1 gene by usingCAN-12v1 sequences as probes or by determining CAN-12v1 protein or mRNAexpression levels. CAN-12v1 polypeptides may be useful for screeningcompounds that affect the activity of the protein. CAN-12v1 peptides canalso be used for the generation of specific antibodies and as bait inyeast two hybrid screens to find proteins the specifically interact withCAN-12v1 (described elsewhere herein).

The CAN-12v1 polynucleotides and polypeptides, including fragments andagonists thereof, may have uses which include detecting, diagnosing,treating, ameliorating, and/or preventing metabolic diseases anddisorders, such as diabetes. Moreover, expressed human CAN-12v1 may beuseful in the detection of patients susceptible to diabetes. Alsoparadigms that would simulate intracellular CAN-12v1 activity would beuseful in treating diabetes.

The CAN-12v1 polynucleotides and polypeptides, including fragmentsthereof, may have uses which include identifying inhibitors ofintracellular calpain inhibitors (calpastatins) leading to an effectiveincrease in calpain activity.

Various approaches to detect alterations or allelic variants at thegenomic or mRNA level of CAN-12v1, could be used as a diagnostic foridentifying MS patients, or individuals susceptible to have MS. It islikely that the CAN-12v1 gene comprises polymorphic sites (i.e. SNPs),with specific alleles which may be associated with MS or otherneurodegenerative disorders, or associated with an increased likelihoodof developing these diseases. Therefore, the invention provides theCAN-12v1 sequence that can be used to design specific primers for theidentification of polymorphisms or mutations in CAN-12v1 of patientsaffected with MS, The presence of a specific allele variant, such as aSNP allele or SNPs haplotype that renders the subject carrying it moresusceptible to develop MS or other related diseases could be identified(e.g. a variant in the CAN-12v1 promoter region that increasedtranscript levels of CAN-i 2v1, or mutations in the coding sequence thatincreased the stability or half-life of the CAN-12v1 protein). Othermethods such as Northern-blot analysis could be performed to measuretranscript levels using a CAN-12v1 cDNA probe derived from the sequenceof the invention.

Although it is believed the encoded polypeptide may share at least somebiological activities with human calpains (particularly m-calpains), anumber of methods of determining the exact biological function of thisclone are either known in the art or are described elsewhere herein.Briefly, the function of this clone may be determined by applyingmicroarray methodology. Nucleic acids corresponding to the CAN-12v1polynucleotides, in addition to, other clones of the present invention,may be arrayed on microchips for expression profiling. Depending onwhich polynucleotide probe is used to hybridize to the slides, a changein expression of a specific gene may provide additional insight into thefunction of this gene based upon the conditions being studied. Forexample, an observed increase or decrease in expression levels when thepolynucleotide probe used comes from diseased neural tissue, as comparedto, normal tissue might indicate a function in modulating neuralfunction, for example. In the case of CAN-12v1, spinal cord, lymph node,thymus, and/or spleen tissue should be used to extract RNA to preparethe probe.

In addition, the function of the protein may be assessed by applyingquantitative PCR methodology, for example. Real time quantitative PCRwould provide the capability of following the expression of the CAN-12v1gene throughout development, for example. Quantitative PCR methodologyrequires only a nominal amount of tissue from each developmentallyimportant step is needed to perform such experiments. Therefore, theapplication of quantitative PCR methodology to refining the biologicalfunction of this polypeptide is encompassed by the present invention. Inthe case of CAN-12v1, a disease correlation related to CAN-12v1 may bemade by comparing the mRNA expression level of CAN-12v1 in normaltissue, as compared to diseased tissue (particularly diseased tissueisolated from the following: esophagus, spinal cord, lymph node, thymus,and/or spleen tissue). Significantly higher or lower levels of CAN-12v1expression in the diseased tissue may suggest CAN-12v1 plays a role indisease progression, and antagonists against CAN-12v1 polypeptides wouldbe useful therapeutically in treating, preventing, and/or amelioratingthe disease. Alternatively, significantly higher or lower levels ofCAN-12v1 expression in the diseased tissue may suggest CAN-12v1 plays adefensive role against disease progression, and agonists of CAN-12v1polypeptides may be useful therapeutically in treating, preventing,and/or ameliorating the disease. Also encompassed by the presentinvention are quantitative PCR probes corresponding to thepolynucleotide sequence provided as SEQ ID NO:53 (FIGS. 8A-C).

The function of the protein may also be assessed through complementationassays in yeast. For example, in the case of the CAN-12v1, transformingyeast deficient in calpain activity, particularly m-calpain activity,and assessing their ability to grow would provide convincing evidencethe CAN-12v1 polypeptide has calpain activity, and possibly m-calpainactivity. Additional assay conditions and methods that may be used inassessing the function of the polynucleotides and polypeptides of thepresent invention are known in the art, some of which are disclosedelsewhere herein.

Alternatively, the biological function of the encoded polypeptide may bedetermined by disrupting a homologue of this polypeptide in Mice and/orrats and observing the resulting phenotype. Such knock-out experimentsare known in the art, some of which are disclosed elsewhere herein.

Moreover, the biological function of this polypeptide may be determinedby the application of antisense and/or sense methodology and theresulting generation of transgenic mice and/or rats. Expressing aparticular gene in either sense or antisense orientation in a transgenicmouse or rat could lead to respectively higher or lower expressionlevels of that particular gene. Altering the endogenous expressionlevels of a gene can lead to the observation of a particular phenotypethat can then be used to derive indications on the function of the gene.The gene can be either over-expressed or under expressed in every cellof the organism at all times using a strong ubiquitous promoter, or itcould be expressed in one or more discrete parts of the organism using awell characterized tissue-specific promoter (e.g., an esophagus, spinalcord, lymph node, thymus, or spleen specific promoter), or it can beexpressed at a specified time of development using an inducible and/or adevelopmentally regulated promoter.

In the case of CAN-12v1 transgenic mice or rats, if no phenotype isapparent in normal growth conditions, observing the organism underdiseased conditions (neural, immune, hematopoietic diseases ordisorders, cancers, etc.) may lead to understanding the function of thegene. Therefore, the application of antisense and/or sense methodologyto the creation of transgenic mice or rats to refine the biologicalfunction of the polypeptide is encompassed by the present invention.

In preferred embodiments, the following N-terminal CAN-12v1 deletionpolypeptides are encompassed by the present invention: M1-L694, S2-L694,L3-L694, W4-L694, P5-L694, P6-L694, F7-L694, R8-L694, C9-L694, R10-L694,W11-L694, K12-L694, L13-L694, A14-L694, P15-L694, R16-L694, Y17-L694,S18-L694, R19-L694, R20-L694, A21-L694, S22-L694, P23-L694, Q24-L694,Q25-L694, P26-L694, Q27-L694, Q28-L694, D29-L694, F30-L694, E31-L694,A32-L694, L33-L694, L34-L694, A35-L694, E36-L694, C37-L694, L38-L694,R39-L694, N40-L694, G41-L694, C42-L694, L43-L694, F44-L694, E45-L694,D46-L694, T47-L694, S48-L694, F49-L694, P50-L694, A51-L694, T52-L694,L53-L694, S54-L694, S55-L694, I56-L694, G57-L694, S58-L694, G59-L694,S60-L694, L61-L694, L62-L694, Q63-L694, K64-L694, L65-L694, P66-L694,P67-L694, R68-L694, L69-L694, Q70-L694, W71-L694, K72-L694, R73-L694,P74-L694, P75-L694, E76-L694, L77-L694, H78-L694, S79-L694, N80-L694,P81-L694, Q82-L694, F83-L694, Y84-L694, F85-L694, A86-L694, K87-L694,A88-L694, K89-L694, R90-L694, L91-L694, D92-L694, L93-L694, C94-L694,Q95-L694, G96-L694, I97-L694, V98-L694, G99-L694, D100-L694, C101-L694,W102-L694, F103-L694, L104-L694, A105-L694, A106-L694, L107-L694,Q108-L694, A109-L694, L110-L694, A111-L694, L112-L694, H113-L694,Q114,-L694, D115-L694, I116-L694, L117-L694, S118-L694, R119-L694,V120-L694, V121-L694, P122-L694, L123-L694, N124-L694, Q125-L694,S126-L694, F127-L694, T128-L694, E129-L694, K130-L694, Y131-L694,A132-L694, G133-L694, I134-L694, F135-L694, R136-L694, F137-L694,W138-L694, F139-L694, W140-L694, H141-L694, Y142-L694, G143-L694,N144-L694, W145-L694, V146-L694, P147-L694, V148-L694, V149-L694,I150-L694, D151-L694, D152-L694, R153-L694, L154-L694, P155-L694,V156-L694, N157-L694, E158-L694, A159-L694, G160-L694, Q161-L694,L162-L694, V163-L694, F164-L694, V165-L694, S166-L694, S167-L694,T168-L694, Y169-L694, K170-L694, N171-L694, L172-L694, F173-L694,W174-L694, G175-L694, A176-L694, L177-L694, L178-L694, E179-L694,K180-L694, A181-L694, Y182-L694, A183-L694, K184-L694, L185-L694,S186-L694, G187-L694, S188-L694, Y189-L694, E190-L694, D191-L694,L192-L694, Q193-L694, S194-L694, G195-L694, Q196-L694, V197-L694,S198-L694, E199-L694, A200-L694, L201-L694, V202-L694, D203-L694,F204-L694, T205-L694, G206-L694, G207-L694, V208-L694, T209-L694,M210-L694, T211-L694, I212-L694, N213-L694, L214-L694, A215-L694,E216-L694, A217-L694, H218-L694, G219-694, N220-L694, L221-L694,W222-L694, D223-L694, 1224-L694, L225-L694, I226-L694, E227-L694,A228-L694, T229-L694, Y230-L694, N231-L694, R232-L694, T233-L694,L234-L694, I235-L694, G236-L694, C237-L694, Q238-L694, T239-L694,H240-L694, S241-L694, G242-L694, E243-L694, K244-L694, I245-L694,L246-L694, E247-L694, N248-L694, G249-L694, L250-L694, V251-L694,E252-L694, G253-L694, H254-L694, A255-L694, Y256-L694, T257-L694,L258-L694, T259-L694, G260-L694, I261-L694, R262-L694, K263-L694,V264-L694, T265-L694, C266-L694, K267-L694, H268-L694, R269-L694,P270-L694, E271-L694, Y272-L694, L273-L694, V274-L694, K275-L694,L276-L694, R277-L694, N278-L694, P279-L694, W280-L694, G281-L694,K282-L694, V283-L694, E284-L694, W285-L694, K286-L694, G287-L694,D288-L694, W289-L694, S290-L694, D291-L694, S292-L694, S293-L694,S294-L694, K295-L694, W296-L694, E297-L694, L298-L694, L299-L694,S300-L694, P301-L694, K302-L694, E303-L694, K304-L694, I305-L694,L306-L694, L307-L694, L308-L694, R309-L694, K310-L694, D311-L694,N312-L694, D313-L694, G314-L694, E315-L694, F316-L694, W317-L694,M318-L694, T319-L694, L320-L694, Q321-L694, D322-L694, F323-L694,K324-L694, T325-L694, H326-L694, F327-L694, V328-L694, L329-L694,L330-L694, V331-L694, I332-L694, C333-L694, K334-L694, L335-L694,T336-L694, P337-L694, G338-L694, L339-L694, L340-L694, S341-L694,Q342-L694, E343-L694, A344-L694, A345-L694, Q346-L694, K347-L694,W348-L694, T349-L694, Y350-L694, T351-L694, M352-L694, R353-L694,E354-L694, G355-L694, R356-L694, W357-L694, E358-L694, K359-L694,R360-L694, S361-L694, T362-L694, A363-L694, G364-L694, G365-L694,Q366-L694, R367-L694, Q368-L694, L369-L694, L370-L694, Q371-L694,D372-L694, T373-L694, F374-L694, W375-L694, K376-L694, N377-L694,P378-L694, Q379-L694, F380-L694, L381-L694, L382-L694, S383-L694,V384-L694, W385-L694, R386-L694, P387-L694, E388-L694, E389-L694,G390-L694, R391-L694, R392-L694, S393-L694, L394-L694, R395-L694,P396-L694, C397-L694, S398-L694, V399-L694, L400-L694, V401-L694,S402-L694, L403-L694, L404-L694, Q405-L694, K406-L694, P407-L694,R408-L694, H409-L694, R410-L694, C411-L694, R412-L694, K413-L694,R414-L694, K415-L694, P416-L694, L417-L694, L418-L694, A419-L694,I420-L694, G421-L694, F422-L694, Y423-L694, L424-L694, Y425-L694,R426-L694, Y427-L694, H428-L694, D429-L694, D430-L694, Q431-L694,R432-L694, R433-L694, L434-L694, P435-L694, P436-L694, E437-L694,F438-L694, F439-L694, Q440-L694, R441-L694, N442-L694, T443-L694,P444-L694, L445-L694, S446-L694, Q447-L694, P448-L694, D449-L694,R450-L694, F451-L694, L452-L694, K453-L694, E454-L694, K455-L694,E456-L694, V457-L694, S458-L694, Q459-L694, E460-L694, L461-L694,C462-L694, L463-L694, E464-L694, P465-L694, G466-L694, T467-L694,Y468-L694, L469-L694, I470-L694, V471-L694, P472-L694, C473-L694,I474-L694, L475-L694, E476-L694, A477-L694, H478-L694, Q479-L694,K480-L694, S481-L694, E482-L694, F483-L694, V484-L694, L485-L694,R486-L694, V487-L694, F488-L694, S489-L694, R490-L694, K491-L694,H492-L694, I493-L694, F494-L694, Y495-L694, E496-L694, I497-L694,G498-L694, S499-L694, N500-L694, S501-L694, G502-L694, V503-L694,V504-L694, F505-L694, S506-L694, K507-L694, E508-L694, I509-L694,E510-L694, D511-L694, Q512-L694, N513-L694, E514-L694, R515-L694,Q516-L694, D517-L694, E518-L694, F519-L694, F520-L694, T521-L694,K522-L694, F523-L694, F524-L694, E525-L694, K526-L694, H527-L694,P528-L694, E529-L694, I530-L694, N531-L694, A532-L694, V533-L694,Q534-L694, L535-L694, Q536-L694, N537-L694, L538-L694, L539-L694,N540-L694, Q541-L694, and/or M542-L694 of SEQ ID NO:54. Polynucleotidesequences encoding these polypeptides are also provided. The presentinvention also encompasses the use of these N-terminal CAN-12v1 deletionpolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein.

In preferred embodiments, the following C-terminal CAN-12v1 deletionpolypeptides are encompassed by the present invention: M1-L694, M1-L693,M1-T692, M1-T691, M1-N690, M1-F689, M1-I688, M1-L687, M1-Y686, M1-S685,M1-T684, M1-S683, M1-H682, M1-A681, M1-V680, M1-G679, M1-G678, M1-D677,M1-I676, M1-N675, M1-A674, M1-L673, M1-1672, M1-V671, M1-P670, M1-V669,M1-I668, M1-M667, M1-F666, M1-F665, M1-T664, M1-P663, M1-T662, M1-S661,M1-Q660, M1-L659, M1-D658, M1-V657, M1-D656, M1-K655, M1-L654, M1-T653,M1-V652, M1-S651, M1-R650, M1-I649, M1-L648, M1-T647, M1-V646, M1-E645,M1-A644, M1-H643, M1-W642, M1-V641, M1-D640, M1-G639, M1-R638, M1-R637,M1-Q636, M1-R635, M1-I634, M1-L633, M1-T632, M1-C631, M1-G630, M1-A629,M1-R628, M1-T627, M1-H626, M1-G625, M1-C624, M1-S623, M1-W622, M1-S621,M1-K620, M1-R619, M1-H618, M1-R617, M1-616, M1-A615, M1-E614, M1-R613,M1-M612, M1-A611, M1-A610, M1-H609, M1-L608, M1-Q607, M1-E606, M1-W605,M1-N604, M1-L603, M1-Y602, M1-G601, M1-S600, M1-G599, M1-R598, M1-D597,M1-Q596, M1-K595, M1-H594, M1-F593, M1-V592, M1-K591, M1-Q590, M1-S589,M1-L588, M1-K587, M1-L586, M1-Q585, M1-K584, M1-W583, M1-L582, M1-D581,M1-R580, M1-F579, M1-E578, M1-Q577, M1-I576, M1-S575, M1-M574, M1-T573,M1-G572, M1-S571, M1-A570, M1-N569, M1-L568, M1-D567, M1-L566, M1-L565,M1-A564, M1-L563, M1-I562, M1-G561, M1-Q560, M1-C559, M1-A558, M1-E557,M1-L556, M1-S555, M1-F554, M1-F553, M1-P552, M1-Q551, M1-R550, M1-S549,M1-G548, M1-L547, M1-S546, M1-S545, M1-W544, M1-T543, M1-M542, M1-Q541,M1-N540, M1-L539, M1-L538, M1-N537, M1-Q536, M1-L535, M1-Q534, M1-V533,M1-A532, M1-N531, M1-1530, M1-E529, M1-P528, M1-H527, M1-K526, M1-E525,M1-F524, M1-F523, M1-K522, M1-T521, M1-F520, M1-F519, M1-E518, M1-D517,M1-Q516, M1-R515, M1-E514, M1-N513, M1-Q512, M1-D511, M1-E510, M1-I509,M1-E508, M1-K507, M1-S506, M1-F505, M1-V504, M1-V503, M1-G502, M1-S501,M1-N500, M1-S499, M1-G498, M1-I497, M1-E496, M1-Y495, M1-F494, M1-1493,M1-H492, M1-K491, M1-R490, M1-S489, M1-F488, M1-V487, M1-R486, M1-L485,M1-V484, M1-F483, M1-E482, M1-S481, M1-K480, M1-Q479, M1-H478, M1-A477,M1-E476, M1-L475, M1-I474, M1-C473, M1-P472, M1-V471, M1-I470, M1-L469,M1-Y468, M1-T467, M1-G466, M1-P465, M1-E464, M1-L463, M1-C462, M1-L461,M1-E460, M1-Q459, M1-S458, M1-V457, M1-E456, M1-K455, M1-E454, M1-K453,M1-L452, M1-F451, M1-R450, M1-D449, M1-P448, M1-Q447, M1-S446, M1-L445,M1-P444, M1-T443, M1-N442, M1-R441, M1-Q440, M1-F439, M1-F438, M1-E437,M1-P436, M1-P435, M1-L434, M1-R433, M1-R432, M1-Q431, M1-D430, M1-D429,M1-H428, M1-Y427, M1-R426, M1-Y425, M1-L424, M1-Y423, M1-F422, M1-G421,M1-1420, M1-A419, M1-L418, M1-L417, M1-P416, M1-K415, M1-R414, M1-K413,M1-R412, M1-C411, M1-R410, M1-H409, M1-R408, M1-P407, M1-K406, M1-Q405,M1-L404, M1-L403, M1-S402, M1-V401, M1-L400, M1-V399, M1-S398, M1-C397,M1-P396, M1-R395, M1-L394, M1-S393, M1-R392, M1-R391, M1-G390, M1-E389,M1-E388, M1-P387, M1-R386, M1-W385, M1-V384, M1-S383, M1-L382, M1-L381,M1-F380, M1-Q379, M1-P378, M1-N377, M1-K376, M1-W375, M1-F374, M1-T373,M1-D372, M1-Q371, M1-L370, M1-L369, M1-Q368, M1-R367, M1-Q366, M1-G365,M1-G364, M1-A363, M1-T362, M1-S361, M1-R360, M1-K359, M1-E358, M1-W357,M1-R356, M1-G355, M1-E354, M1-R353, M1-M352, M1-T351, M1-Y350, M1-T349,M1-W348, M1-K347, M1-Q346, M1-A345, M1-A344, M1-E343, M1-Q342, M1-S341,M1-L340, M1-L339, M1-G338, M1-P337, M1-T336, M1-L335, M1-K334, M1-C333,M1-I332, M1-V331, M1-L330, M1-L329, M1-V328, M1-F327, M1-H326, M1-T325,M1-K324, M1-F323, M1-D322, M1-Q321, M1-L320, M1-T319, M1-M318, M1-W317,M1-F316, M1-E315, M1-G314, M1-D313, M1-N312, M1-D311, M1-K310, M1-R309,M1-L308, M1-L307, M1-L306, M1-I305, M1-K304, M1-E303, M1-K302, M1-P301,M1-S300, M1-L299, M1-L298, M1-E297, M1-W296, M1-K295, M1-S294, M1-S293,M1-S292, M1-D291, M1-S290, M1-W289, M1-D288, M1-G287, M1-K286, M1-W285,M1-E284, M1-V283, M1-K282, M1-G281, M1-W280, M1-P279, M1-N278, M1-R277,M1-L276, M1-K275, M1-V274, M1-L273, M1-Y272, M1-E271, M1-P270, M1-R269,M1-H268, M1-K267, M1-C266, M1-T265, M1-V264, M1-K263, M1-R262, M1-I261,M1-G260, M1-T259, M1-L258, M1-T257, M1-Y256, M1-A255, M1-H254, M1-G253,M1-E252, M1-V251, M1-L250, M1-G249, M1-N248, M1-E247, M1-L246, M1-I245,M1-K244, and/or M1-E243 of SEQ ID NO:54. Polynucleotide sequencesencoding these polypeptides are also provided. The present inventionalso encompasses the use of these C-terminal CAN-12v1 deletionpolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein.

Alternatively, preferred polypeptides of the present invention maycomprise polypeptide sequences corresponding to, for example, internalregions of the CAN-12v1 polypeptide (e.g., any combination of both N—and C-terminal CAN-12v1 polypeptide deletions) of SEQ ID NO:54. Forexample, internal regions could be defined by the equation: amino acidNX to amino acid CX, wherein NX refers to any N-terminal deletionpolypeptide amino acid of CAN-12v1 (SEQ ID NO:54), and where CX refersto any C-terminal deletion polypeptide amino acid of CAN-12v1 (SEQ IDNO:54). Polynucleotides encoding these polypeptides are also provided.The present invention also encompasses the use of these polypeptides asan immunogenic and/or antigenic epitope as described elsewhere herein.

The present invention also encompasses immunogenic and/or antigenicepitopes of the CAN-12v1 polypeptide.

The CAN-12v1 polypeptides of the present invention were determined tocomprise several phosphorylation sites based upon the Motif algorithm(Genetics Computer Group, Inc.). The phosphorylation of such sites mayregulate some biological activity of the CAN-12v1 polypeptide. Forexample, phosphorylation at specific sites may be involved in regulatingthe proteins ability to associate or bind to other molecules (e.g.,proteins, ligands, substrates, DNA, etc.). In the present case,phosphorylation may modulate the ability of the CAN-12v1 polypeptide toassociate with other polypeptides, particularly the serine proteasesubstrate for CAN-12v1, or its ability to modulate serine proteasefunction.

The CAN-12v1 polypeptide was predicted to comprise eleven PKCphosphorylation sites using the Motif algorithm (Genetics ComputerGroup, Inc.). In vivo, protein kinase C exhibits a preference for thephosphorylation of serine or threonine residues. The PKC phosphorylationsites have the following consensus pattern: [ST]-x-[RK], where S or Trepresents the site of phosphorylation and ‘x’ an intervening amino acidresidue. Additional information regarding PKC phosphorylation sites canbe found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem.161:177-184(1986), and Kishimoto A., Nishiyama K., Nakanishi H.,Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem.260:12492-12499(1985); which are hereby incorporated by referenceherein.

In preferred embodiments, the following PKC phosphorylation sitepolypeptides are encompassed by the present invention: LAPRYSRRASPQQ(SEQ ID NO:58), LNQSFTEKYAGIF (SEQ ID NO:59), VFVSSTYKNLFWG (SEQ IDNO:60), GIRKVTCKHRPEY (SEQ ID NO:61), DWSDSSSKWELLS (SEQ ID NO:62),KWELLSPKEKILL (SEQ ID NO:63), QKWTYTMREGRWE (SEQ ID NO:64),EEGRRSLRPCSVL (SEQ ID NO:65), VLRVFSRKHIFYE (SEQ ID NO:66),KQLKLSQKVFHKQ (SEQ ID NO:67), and/or LIRSVTLKDVDLQ (SEQ ID NO:68).Polynucleotides encoding these polypeptides are also provided. Thepresent invention also encompasses the use of the CAN-12v1 PKCphosphorylation site polypeptides as immunogenic and/or antigenicepitopes as described elsewhere herein.

The CAN-12v1 polypeptide has been shown to comprise four glycosylationsite according to the Motif algorithm (Genetics Computer Group, Inc.).As discussed more specifically herein, protein glycosylation is thoughtto serve a variety of functions including: augmentation of proteinfolding, inhibition of protein aggregation, regulation of intracellulartrafficking to organelles, increasing resistance to proteolysis,modulation of protein antigenicity, and mediation of intercellularadhesion.

In preferred embodiments, the following asparagine glycosylation sitepolypeptide is encompassed by the present invention: RVVPLNQSFTEKYA (SEQID NO:69), IEATYNRTLIGCQT (SEQ ID NO:70), ALLDLNASGTMSIQ (SEQ ID NO:71),and/or SYLIFNTTLL (SEQ ID NO:72). Polynucleotides encoding thispolypeptide are also provided. The present invention also encompassesthe use of the CAN-12v1 asparagine glycosylation site polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

The CAN-12v1 polypeptide has been shown to comprise one amidation siteaccording to the Motif algorithm (Genetics Computer Group, Inc.). Theprecursor of hormones and other active peptides which are C-terminallyamidated is always directly followed by a glycine residue which providesthe amide group, and most often by at least two consecutive basicresidues (Arg or Lys) which generally function as an active peptideprecursor cleavage site. Although all amino acids can be amidated,neutral hydrophobic residues such as Val or Phe are good substrates,while charged residues such as Asp or Arg are much less reactive. Aconsensus pattern for amidation sites is the following: x-G-[RK]-[RK],wherein “X” represents the amidation site. Additional informationrelating to asparagine glycosylation may be found in reference to thefollowing publications, which are hereby incorporated by referenceherein: Kreil G., Meth. Enzymol. 106:218-223(1984); and Bradbury A. F.,Smyth D. G., Biosci. Rep. 7:907-916(1987).

In preferred embodiments, the following amidation site polypeptide isencompassed by the present invention: VWRPEEGRRSLRPC (SEQ ID NO:73).Polynucleotides encoding these polypeptides are also provided. Thepresent invention also encompasses the use of this CAN-12v1 amidationsite polypeptide as an immunogenic and/or antigenic epitope as describedelsewhere herein.

The CAN-12v1 polypeptide has been shown to comprise one RGD cellattachment site domain according to the Motif algorithm (GeneticsComputer Group, Inc.). The sequence Arg-Gly-Asp, found in fibronectin,is crucial for its interaction with its cell surface receptor, anintegrin. What has been called the ‘RGD’ tripeptide is also found in thesequences of a number of other proteins, where it has been shown to playa role in cell adhesion. Non-limiting examples of these proteins are thefollowing: some forms of collagens, fibrinogen, vitronectin, vonWillebrand factor (VWF), snake disintegrins, and slime mold discoidins.The ‘RGD’ tripeptide is also found in other proteins where it may servethe same purpose. A consensus pattern for RGD cell attachment sites isthe following: R-G-D. Additional information relating to RGD cellattachment site domains may be found in reference to the followingpublications, which are hereby incorporated by reference herein:Ruoslahti E., Pierschbacher M. D., Cell 44:517-518(1986); and d'Souza S.E., Ginsberg M. H., Plow E. F., Trends Biochem. Sci. 16:246-250(1991).

In preferred embodiments, the following RGD cell attachment site domainpolypeptide is encompassed by the present invention: LIRQRRGDVWHAE (SEQID NO:74). Polynucleotides encoding these polypeptides are alsoprovided. The present invention also encompasses the use of this RGDcell attachment site domain polypeptide as an immunogenic and/orantigenic epitope as described elsewhere herein.

In confirmation of the CAN-12v1 polypeptide being a calpain, it has beenshown to comprise one EF-hand calcium-binding domain according to theMotif algorithm (Genetics Computer Group, Inc.). Many calcium-bindingproteins belong to the same evolutionary family and share a type ofcalcium-binding domain known as the EF-hand. This type of domainconsists of a twelve residue loop flanked on both side by a twelveresidue alpha-helical domain. In an EF-hand loop the calcium ion iscoordinated in a pentagonal bipyramidal configuration. The six residuesinvolved in the binding are in positions 1, 3, 5, 7, 9 and 12; theseresidues are denoted by X, Y, Z, —Y, —X and -Z. The invariant Glu or Aspat position 12 provides two oxygens for liganding Ca (bidentate ligand).Several representative proteins containing EF-hand regions are providedbelow: For each type of protein, the total number of EF-hand regionsknown or supposed to exist are provided in parenthesis: Aequorin andRenilla luciferin binding protein (LBP) (Ca=3); Alpha actinin (Ca=2);Calbindin (Ca=4); Calcineurin B subunit (protein phosphatase 2Bregulatory subunit) (Ca=4); Calcium-binding protein from Streptomyceserythraeus (Ca=3?); Calcium-binding protein from Schistosoma mansoni(Ca=2?); Calcium-binding proteins TCBP-23 and TCBP-25 from Tetrahymenathermophila (Ca=4?); Calcium-dependent protein kinases (CDPK) fromplants (Ca=4); Calcium vector protein from amphoxius (Ca=2); Calcyphosin(thyroid protein p24) (Ca=4?); Calmodulin (Ca=4, except in yeast whereCa=3); Calpain small and large chains (Ca=2); Calretinin (Ca=6);Calcyclin (prolactin receptor associated protein) (Ca=2); Caltractin(centrin) (Ca=2 or 4); Cell Division Control protein 31 (gene CDC31)from yeast (Ca=2?); Diacylglycerol kinase (EC 2.7.1.107) (DGK) (Ca=2);FAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) frommammals (Ca=1); Fimbrin (plastin) (Ca=2); Flagellar calcium-bindingprotein (1f8) from Trypanosoma cruzi (Ca=1 or 2); Guanylate cyclaseactivating protein (GCAP) (Ca=3); Inositol phospholipid-specificphospholipase C isozymes gamma-1 and delta-1 (Ca=2) [10]; Intestinalcalcium-binding protein (ICaBPs) (Ca=2); MIF related proteins 8 (MRP-8or CFAG) and 14 (MRP-14) (Ca=2); Myosin regulatory light chains (Ca=1);Oncomodulin (Ca=2); Osteonectin (basement membrane protein BM-40)(SPARC) and proteins that contains an ‘osteonectin’ domain (QR1, matrixglycoprotein SC1) (Ca=1); Parvalbumins alpha and beta (Ca=2); Placentalcalcium-binding protein (18a2) (nerve growth factor induced protein 42a)(p9k) (Ca=2); Recoverins (visinin, hippocalcin, neurocalcin, S-modulin)(Ca=2 to 3); Reticulocalbin (Ca=4); S-100 protein, alpha and beta chains(Ca=2); Sarcoplasmic calcium-binding protein (SCPs) (Ca=2 to 3); Seaurchin proteins Spec 1 (Ca=4), Spec 2 (Ca=4?), Lps-1 (Ca=8);Serine/threonine protein phosphatase rdgc (EC 3.1.3.16) from Drosophila(Ca=2); Sorcin V19 from hamster (Ca=2); Spectrin alpha chain (Ca-2);Squidulin (optic lobe calcium-binding protein) from squid (Ca=4); andTroponins C; from skeletal muscle (Ca=4), from cardiac muscle (Ca=3),from arthropods and molluscs (Ca=2).

A consensus pattern for EF hand calcium binding domains is thefollowing:  1 2 3  4     5  6     7  8   9     10  12   13 X   Y        Z       -Y     -X    -ZD-x-[DNS]-{ILVFYW}-[DENSTG]-[DNQGHRK]-{GP}-[LIVMC]-[DENQSTAGC]-x(2)-[DE]-[LIVMFYW],wherein X, Y, Z, —Y, —X, and -Z are as defined above, and wherein “x”represents any amino acid. Amino acid residues within the consensus atpositions 1 (X), 3 (Y) and 12 (-Z) are the most conserved. The 6thresidue in an EF-hand loop is in most cases a Gly.

Additional information relating to RGD cell attachment site domains maybe found in reference to the following publications, which are herebyincorporated by reference herein: Kawasaki H., Kretsinger R. H., ProteinProf. 2:305-490(1995); Kretsinger R. H., Cold Spring Harbor Symp. Quant.Biol. 52:499-510(1987); Moncrief N. D., Kretsinger R. H., Goodman M., J.Mol. Evol. 30:522-562(1990); Nakayama S., Moncrief N. D., Kretsinger R.H., J. Mol. Evol. 34:416-448(1992); Heizmann C. W., Hunziker W., TrendsBiochem. Sci. 16:98-103(1991); Kligman D., Hilt D. C., Trends Biochem.Sci. 13:437-443(1988); Strynadka N. C. J., James M. N. G., Annu. Rev.Biochem. 58:951-98(1989); Haiech J., Sallantin J., Biochimie67:555-560(1985); Chauvaux S., Beguin P., Aubert J.-P., Bhat K. M., GowL. A., Wood T. M., Bairoch A., Biochem. J. 265:261-265(1990); BairochA., Cox J. A., FEBS Lett. 269:454-456(1990).

In preferred embodiments, the following EF-hand calcium binding domainpolypeptide is encompassed by the present invention:WLALLDLNASGTMSIQEFRDLWK (SEQ ID NO:75). Polynucleotides encoding thesepolypeptides are also provided. The present invention also encompassesthe use of this EF-hand calcium binding domain polypeptide as animmunogenic and/or antigenic epitope as described elsewhere herein.

In further confirmation of the CAN-12v1 polypeptide being a calpain, ithas been shown to comprise one eukaryotic thiol (cysteine) proteaseactive site domain according to the Motif algorithm (Genetics ComputerGroup, Inc.). Eukaryotic thiol proteases (EC 3.4.22.-) are a family ofproteolytic enzymes which contain an active site cysteine. Catalysisproceeds through a thioester intermediate and is facilitated by a nearbyhistidine side chain; an asparagine completes the essential catalytictriad. Non-limiting examples of proteases which are known to belong tothis family are provided below: Vertebrate lysosomal cathepsins B (EC3.4.22.1), H (EC 3.4.22.16), L (EC 3.4.22.15), and S (EC 3.4.22.27);Vertebrate lysosomal dipeptidyl peptidase I (EC 3.4.14.1) (also known ascathepsin C); Vertebrate calpains (EC 3.4.22.17) (Calpains areintracellular calcium-activated thiol protease that contain both aN-terminal catalytic domain and a C-terminal calcium-binding domain;Mammalian cathepsin K, which seems involved in osteoclastic boneresorption; Human cathepsin O; Bleomycin hydrolase (An enzyme thatcatalyzes the inactivation of the antitumor drug BLM (a glycopeptide);Plant enzymes: barley aleurain (EC 3.4.22.16), EP-B1/B4; kidney beanEP-C1, rice bean SH-EP; kiwi fruit actinidin (EC 3.4.22.14); papayalatex papain (EC 3.4.22.2), chymopapain (EC 3.4.22.6), caricain (EC3.4.22.30), and proteinase IV (EC 3.4.22.25); pea turgor-responsiveprotein 15A; pineapple stem bromelain (EC 3.4.22.32); rape COT44; riceoryzain alpha, beta, and gamma; tomato low-temperature induced,Arabidopsis thaliana A494, RD19A and RD21A; House-dust mites allergensDerP1 and EurM1; Cathepsin B-like proteinases from the wormsCaenorhabditis elegans (genes gcp-1, cpr-3, cpr-4, cpr-5 and cpr-6),Schistosoma mansoni (antigen SM31) and Japonica (antigen SJ3 1),Haemonchus contortus (genes AC-1 and AC-2), and Ostertagia ostertagi(CP-1 and CP-3); Slime mold cysteine proteinases CP1 and CP2; Cruzipainfrom Trypanosoma cruzi and brucei; Throphozoite cysteine proteinase(TCP) from various Plasmodium species; Proteases from Leishmaniamexicana, Theileria annulata and Theileria parva; Baculovirusescathepsin-like enzyme (v-cath); Drosophila small optic lobes protein(gene sol), a neuronal protein that contains a calpain-like domain;Yeast thiol protease BLH1/YCP1/LAP3; and Caenorhabditis eleganshypothetical protein C06G4.2, a calpain-like protein; Two bacterialpeptidases are also part of this family—Aminopeptidase C fromLactococcus lactis (gene pepC), and Thiol protease tpr fromPorphyromonas gingivalis.

A consensus pattern for eukaryotic thiol (cysteine) protease active sitedomains is the following: Q-x(3)-[GE]-x-C-[YW]-x(2)-[STAGC]-[STAGCV],wherein C is the active site residue, and “x” represents any amino acid.The residue in position 4 of the pattern is almost always cysteine; theonly exceptions are calpains (Leu), bleomycin hydrolase (Ser) and yeastYCP1 (Ser); while the residue in position 5 of the pattern is always Glyexcept in papaya protease IV where it is Glu.

An additional consensus pattern for eukaryotic thiol (cysteine) proteaseactive site domains is the following:[LIVMGSTAN]-x-H-[GSACE]-[LIVM]-x-[LIVMAT](2)-G-x-[GSADNH], wherein H isthe active site residue, and “x” represents any amino acid.

An additional consensus pattern for eukaryotic thiol (cysteine) proteaseactive site domains is the following:[FYCH]-[WI]-[LIVT]-x-[KRQAG]-N-[ST]-W-x(3)-[FYW]-G-x(2)-G-[LFYW]-[LIVMFYG]-x-[LIVMF],wherein N is the active site residue, and “x” represents any amino acid.

Additional information relating to for eukaryotic thiol (cysteine)protease active site domains may be found in reference to the followingpublications, which are hereby incorporated by reference herein: DufourE., Biochimie 70:1335-1342(1988); Kirschke H., Barrett A. J., RawlingsN. D., Protein Prof. 2:1587-1643(1995); Shi G.-P., Chapman H. A., BhairiS. M., Deleeuw C., Reddy V. Y., Weiss S. J., FEBS Lett.357:129-134(1995); Velasco G., Ferrando A. A., Puente X. S., Sanchez L.M., Lopez-Otin C., J. Biol. Chem. 269:27136-27142(1994); Chapot-ChartierM. P., Nardi M., Chopin M. C., Chopin A., Gripon J. C., Appl. Environ.Microbiol. 59:330-333(1993); Higgins D. G., McConnell D. J., Sharp P.M., Nature 340:604-604(1989); Rawlings N. D., Barrett A. J., Meth.Enzymol. 244:461-486(1994), andhttp://www.expasy.ch/cgi-bin/lists?peptidas.txt, which are herebyincorporated by reference in their entirety herein.

In preferred embodiments, the following for eukaryotic thiol (cysteine)protease active site domain polypeptide is encompassed by the presentinvention: RLDLCQGIVGDCWFLAALQALA (SEQ ID NO:76). Polynucleotidesencoding these polypeptides are also provided. The present inventionalso encompasses the use of this for eukaryotic thiol (cysteine)protease active site domain polypeptide as an immunogenic and/orantigenic epitope as described elsewhere herein.

As referenced elsewhere herein, calpains are organized in domains. As apoint of reference, the larger catalytic subunit of the bestcharacterized m-calpain is organized in four domains (I-IV)(Hosfield etal., Crystal structure of calpain reveals the structural basis forCa(2+)-dependent protease activity and a novel mode of enzymeactivation. EMBO J 18:6880-9, 1999; Strobl et al., The crystal structureof calcium-free human m-calpain suggests an electrostatic switchmechanism for activation by calcium. Proc Natl Acad Sci U S A.97:588-92, 2000). The N-termninal domain I contains an alpha helicalregion. Domain II contains the catalytic active domain with the activesite amino acids. Domain III contains the linker between the Ca2+binding domain in domain IV to the active site domain II.

The CAN-12v1 calpain of the present invention has the same domain I andII as the CAN-12 calpain, but differ in domains III and IV. TheN-terminal domain I consists of residues Met1-Arg20. Domain II of thepresent calpains (Ala21-Lys333) contain the catalytic active siteresidues acids (Cys101, His254 and Asn 278). As can be seen in thesequence alignments (FIGS. 2A-E), there is high amino acid sequencehomology in the amino acid residues bracketing the active site aminoacids. Combined domains I and II of the calpains of the presentinvention are 42-45% homologous to m-calpain.

The CAN-12v1 calpain of the present invention, have the same domain Iand II, although they differ in composition and content of domains IIIand IV. The CAN-12 and CAN-12v1 calpain contains the linker (domain III)and the C-ternminal domain IV, though the CAN-12v1 calpain is lackingresidues Met426, Asn427 and Lys428 of SEQ ID NO54) in the “linker”domain.

The present invention also provides a three-dimensional homology modelof the CAN-12 polypeptide (see FIG. 6). Although the CAN-12 polypeptidesequence is different than the CAN-12v1 polypeptide sequence, the factthat domain I and II are substantially the same suggests the homologymodel of CAN-12 may be used for designing potential ligands (includingagonists and/or antagonists) for the CAN-12v1 polypeptide. Athree-dimensional homology model can be constructed on the basis of theknown structure of a homologous protein (Greer et al, 1991, Lesk, et al,1992, Cardozo, et al, 1995, Yuan, et al, 1995). The homology model ofthe CAN-12 polypeptide, corresponding to amino acid residues 12 to 524of SEQ ID NO:2, was based upon the homologous structure of CAN2, am-calpain family member (Strobl et al, 2000; hCAN2; Genbank AccessionNo. gi|7546423; SEQ ID NO:11) and is defined by the set of structuralcoordinates set forth in Table IV herein.

A description of the headings in Table IV are as follows: “Atom No”refers to the atom number within the CAN-12 homology model; “Atom name”refers to the element whose coordinates are measured, the first letterin the column defines the element; “Residue” refers to the amino acidwithin which the atom resides, and the provided number after the aminoacid refers to the amino acid number of the “residue”; “X Coord”, “YCoord”, and “Z Coord” structurally define the atomic position of theelement measured in three dimensions.

The CAN-12 homology model of the present invention may provide one basisfor designing rational stimulators (agonists) and/or inhibitors(antagonists) of one or more of the biological functions of CAN-12v1, orof CAN-12v1 mutants having altered specificity (e.g., molecularlyevolved CAN-12v1 polypeptides, engineered site-specific CAN-12v1mutants, CAN-12v1 allelic variants, etc.).

Homology models are not only useful for designing rational agonistsand/or antagonists, but are also useful in predicting the function of aparticular polypeptide. The functional predictions from homology modelsare typically more accurate than the functional attributes derived fromtraditional polypeptide sequence homology alignments (e.g., CLUSTALW),particularly when the three dimensional structure of a relatedpolypeptide is known (e.g., m-calpain family member CAN2 protein;Genbank Accession No. gi|7546423; SEQ ID NO:11). The increasedprediction accuracy is based upon the fact that homology modelsapproximate the three-dimensional structure of a protein, while homologybased alignments only take into account the one dimension polypeptidesequence. Since the function of a particular polypeptide is determinednot only by its primary, secondary, and tertiary structure, functionalassignments derived solely upon homology alignments using the onedimensional protein sequence may be less reliable. A 3-dimensional modelcan be constructed on the basis of the known structure of a homologousprotein (Greer et al, 1991, Lesk, et al, 1992, Cardozo, et al, 1995,Yuan, et al, 1995).

Prior to developing a homology model, those of skill in the art wouldappreciate that a template of a known protein, or model protein, mustfirst be identified which will be used as a basis for constructing thehomology model for the protein of unknown structure (query template). Inthe case of the CAN-12 polypeptide of the present invention, the modelprotein template used in constructing the CAN-12 homology model was them-calpain family member CAN2 protein; Genbank Accession No. gi|7546423;SEQ ID NO:11).

Identifying a template can be accomplished using pairwise alignment ofprotein sequences using such programs as FASTA (Pearson, et al 1990) andBLAST (Altschul, et al, 1990). In cases where sequence similarity ishigh (greater than 30%), such pairwise comparison methods may beadequate for identifying an appropriate template. Likewise, multiplesequence alignments or profile-based methods can be used to align aquery sequence to an alignment of multiple (structurally andbiochemically) related proteins. When the sequence similarity is low,more advanced techniques may be used. Such techniques, include, forexample, protein fold recognition (protein threading; Hendlich, et al,1990), where the compatibility of a particular polypeptide sequence withthe 3-dimensional fold of a potential template protein is gauged on thebasis of a knowledge-based potential.

Following the initial sequence alignment, the second step would be tooptimally align the query template to the model template by manualmanipulation and/or by the incorporation of features specific to thepolypeptides (e.g., motifs, secondary structure predictions, and allowedconservations). Preferably, the incorporated features are found withinboth the model and query template.

The third step would be to identify structurally conserved regions thatcould be used to construct secondary core structure (Sali, et al, 1995).Loops could be added using knowledge-based techniques, and by performingforcefield calculations (Sali, et al, 1995).

The term “structure coordinates” refers to Cartesian coordinatesgenerated from the building of a homology model. In this invention, thehomology model of residues 12 to 524 of CAN-12 was derived fromgenerating a sequence alignment with m-calpain (Strobl et al, 2000;hCAN2; Genbank Accession No. gi|7546423; SEQ ID NO:11) using theCOMPOSER suite of software within SYBYL6.6 (Tripos Associates, St.Louis, Mo.) and then generating the backbone and side chainconformations. In the original crystal structure (pdb code 1dkv) as wellas the crystal structure reported elsewhere (Hosfield et al, 1999), theactive site of the enzyme comprising a cysteine, a histidine and anasparagine residue was not “formed”. The helix that contains the activesite C101 was altered by moving the helix down one pitch so that theactive site geometry could match that found in Papain (pdb code 1b4).This modified structure of human m-calpain was used as the template forconstruction of the homology model (illustrated in FIG. 6 herein).

The skilled artisan would appreciate that a set of structure coordinatesfor a protein represents a relative set of points that define a shape inthree dimensions. Thus, it is possible that an entirely different set ofcoordinates could define a similar or identical shape. Moreover, slightvariations in the individual coordinates, as emanate from the generationof similar homology models using different alignment templates (i.e.,other than the m-calpain (Strobl et al, 2000; hCAN2; Genbank AccessionNo. gi|7546423; SEQ ID NO:11), and/or using different methods ingenerating the homology model, will likely have minor effects on theoverall shape. Variations in coordinates may also be generated becauseof mathematical manipulations of the structure coordinates. For example,the structure coordinates set forth in Table IV could be manipulated byfractionalization of the structure coordinates; integer additions, orinteger subtractions to sets of the structure coordinates, inversion ofthe structure coordinates or any combination of the above.

Therefore, various computational analyses are necessary to determinewhether a template molecule or a portion thereof is sufficiently similarto all or part of a query template (e.g., CAN-12) in order to beconsidered the same. Such analyses may be carried out in currentsoftware applications, such as SYBYL version 6.6 or INSIGHTII (MolecularSimulations Inc., San Diego, Calif.) version 2000 and as described inthe accompanying User's Guides.

Using the superimposition tool in the program SYBYL, comparisons can bemade between different structures and different conformations of thesame structure. The procedure used in SYBYL to compare structures isdivided into four steps: 1) load the structures to be compared; 2)define the atom equivalencies in these structures; 3) perform a fittingoperation; and 4) analyze the results. Each structure is identified by aname. One structure is identified as the target (i.e., the fixedstructure); the second structure (i.e., moving structure) is identifiedas the source structure. The atom equivalency within SYBYL is defined byuser input. For the purpose of this invention, we will define equivalentatoms as protein backbone atoms (N, Cα, C and O) for all conservedresidues between the two structures being compared. We will alsoconsider only rigid fitting operations. When a rigid fitting method isused, the working structure is translated and rotated to obtain anoptimum fit with the target structure. The fitting operation uses analgorithm that computes the optimum translation and rotation to beapplied to the moving structure, such that the root mean squaredifference of the fit over the specified pairs of equivalent atoms is anabsolute minimum. This number, given in angstroms, is reported by theSYBYL program. For the purpose of the present invention, any homologymodel of a CAN-12 that has a root mean square deviation of conservedresidue backbone atoms (N, Cα, C, O) of less than 3.0 A whensuperimposed on the relevant backbone atoms described by structurecoordinates listed in Table IV are considered identical. Morepreferably, the root mean square deviation for the CAN-12 polypeptide isless than 2.0 Å.

The homology model of the present invention is useful for thestructure-based design of modulators of the CAN-12 biological function,as well as mutants with altered biological function and/or specificity.

In accordance with the structural coordinates provided in Table IV andthe three dimensional homology model of CAN-12, the CAN-12v1 polypeptidehas been shown to comprise a an active site region embodied by thefollowing amino acids: from about amino acid R90 to about amino acidC94, from about amino acid G99 to about amino acid L104, from aboutamino acid Q196 to about amino acid S198, from about amino acid M210 toabout amino acid 1212, from about amino acid G236 to about amino acidH240, from about amino acid E252 to about amino acid Y256, from aboutamino acid N278 to about amino acid K282, from about amino acid V328 toabout amino acid V331 of SEQ ID NO:54 (FIGS. 8A-C). In this context, theterm “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acids more in either the N— or C-terminal direction of the abovereferenced amino acids.

Also more preferred are polypeptides comprising all or any part of theCAN-12v1 active site domain, or a mutant or homologue of saidpolypeptide or molecular complex. By mutant or homologue of the moleculeis meant a molecule that has a root mean square deviation from thebackbone atoms of said CAN-12 amino acids of not more than about 4.5Angstroms, and preferably not more than about 3.5 Angstroms.

In preferred embodiments, the following CAN-12v1 active site domainpolypeptide is encompassed by the present invention:RLDLCQGIVGDCWFLAALQALALHQDILSRVVPLNQSFTEKYAGIFRFWFWHYGNWVPVVIDDRLPVNEAGQLVFVSSTYKNLFWGALLEKAYAKLSGSYEDLQSGQVSEALVDFTGGVTMTINLAEAHGNLWDILEATYNRTLIGCQTHSGKILENGLVEGHAYTLTGIRKVTCKHRPEYLVKLRNPWGKVEWKGDWSDSSSKWELLSPKEKILLLRKDNDGEFWMTLQDFKTHFVLLV (SEQ ID NO:92). Polynucleotides encoding thispolypeptide are also provided. The present invention also encompassesthe use of the CAN-12v1 active site domain polypeptide as an immunogenicand/or antigenic epitope as described elsewhere herein.

The present invention also encompasses polypeptides comprising at leasta portion of the CAN-12 active site domain (SEQ ID NO: 92). Suchpolypeptides may correspond, for example, to the N— and/or C-terminaldeletions of the active site domain.

In preferred embodiments, the following N-terminal CAN-12v1 active sitedomain deletion polypeptides are encompassed by the present invention:R1-V242, L2-V242, D3-V242, L4-V242, C5-V242, Q6-V242, G7-V242, I8-V242,V9-V242, G10-V242, D11-V242, C12-V242, W13-V242, F14-V242, L15-V242,A16-V242, A17-V242, L18-V242, Q19- V242, A20-V242, L21-V242, A22-V242,L23-V242, H24-V242, Q25-V242, D26-V242, I27-V242, L28-V242, S29-V242,R30-V242, V31-V242, V32-V242, P33-V242, L34-V242, N35-V242, Q36-V242,S37-V242, F38-V242, T39-V242, E40-V242, K41-V242, Y42-V242, A43-V242,G44-V242, I45-V242, F46-V242, R47-V242, F48-V242, W49-V242, F50-V242,W51-V242, H52-V242, Y53-V242, G54-V242, N55-V242, W56-V242, V57-V242,P58-V242, V59-V242, V60-V242, I61-V242, D62-V242, D63-V242, R64-V242,L65-V242, P66-V242, V67-V242, N68-V242, E69-V242, A70-V242, G71-V242,Q72-V242, L73-V242, V74-V242, F75-V242, V76-V242, S77-V242, S78-V242,T79-V242, Y80-V242, K81-V242, N82-V242, L83-V242, F84-V242, W85-V242,G86-V242, A87-V242, L88-V242, L89-V242, E90-V242, K91-V242, A92-V242,Y93-V242, A94-V242, K95-V242, L96-V242, S97-V242, G98-V242, S99-V242,Y100-V242, E101-V242, D102-V242, L103-V242, Q104-V242, S105-V242,G106-V242, Q107-V242, V108-V242, S109-V242, E110-V242, A111-V242,L112-V242, V113-V242, D114-V242, F115-V242, T116-V242, G117-V242,G118-V242, V119-V242, T120-V242, M121-V242, T122-V242, I123-V242,N124-V242, L125-V242, A126-V242, E127-V242, A128-V242, H129-V242,G130-V242, N131-V242, L132-V242, W133-V242, D134-V242, I135-V242,L136-V242, I137-V242, E138-V242, A139-V242, T140-V242, Y141-V242,N142-V242, R143-V242, T144-V242, L145-V242, I146-V242, G147-V242,C148-V242, Q149-V242, T150-V242, H151-V242, S152-V242, G153-V242,E154-V242, K155-V242, I156-V242, L157-V242, E158-V242, N159-V242,G160-V242, L161-V242, V162-V242, E163-V242, G164-V242, H165-V242,A166-V242, Y167-V242, T168-V242, L169-V242, T170-V242, G171-V242,I172-V242, R173-V242, K174-V242, V175-V242, T176-V242, C177-V242,K178-V242, H179-V242, R180-V242, P181-V242, E182-V242, Y183-V242,L184-V242, V185-V242, K186-V242, L187-V242, R188-V242, N189-V242,P190-V242, W191-V242, G192-V242, K193-V242, V194-V242, E195-V242,W196-V242, K197-V242, G198-V242, D199-V242, W200-V242, S201-V242,D202-V242, S203-V242, S204-V242, S205-V242, K206-V242, W207-V242,E208-V242, L209-V242, L210-V242, S211-V242, P212-V242, K213-V242,E214-V242, K215-V242, I216-V242, L217-V242, L218-V242, L219-V242,R220-V242, K221-V242, D222-V242, N223-V242, D224-V242, G225-V242,E226-V242, F227-V242, W228-V242, M229-V242, T230-V242, L231 -V242,Q232-V242, D233-V242, F234-V242, K235-V242, and/or T236-V242 of SEQ IDNO:92. Polynucleotide sequences encoding these polypeptides are alsoprovided. The present invention also encompasses the use of theseN-terminal CAN-12v1 active site domain deletion polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal CAN-12v1 active sitedomain deletion polypeptides are encompassed by the present invention:R1-V242, R1-L241, R1-L240, R1-V239, R1-F238, R1-H237, R1-T236, R1-K235,R1-F234, R 1-D233, R1-Q232, R1-L231, R1-T230, R1-M229, R1-W228, R1-F227,R1-E226, R1-G225, R1-D224, R1-N223, R1-D222, R1-K221, R1-R220, R1-L219,R1-L218, R1-L217, R1-I216, R1-K215, R1-E214, R1-K213, R1-P212, R1-S211,R1-L210, R1-L209, R1-E208, R1-W207, R1-K206, R1-S205, R1-S204, R1-S203,R1-D202, R1-S201, R1-W200, R1-D199, R1-G198, R1-K197, R1-W196, R1-E195,R1-V194, R1-K193, R1-G192, R1-W191, R1-P190, R1-N189, R1-R188, R1-L187,R1-K186, R1-V185, R1-L184, R1-Y183, R1-E182, R1-P181, R1-R180, R1-H179,R1-K178, R1-C177, R1-T176, R1-V175, R1-K174, R1-R173, R1-I172, R1-G171,R1-T170, R1-L169, R1-T168, R1-Y167, R1-A166, R1-H165, R1-G164, R1-E163,R1-V162, R1-L161, R1-G160, R1-N159, R1-E158, R1-L157, R1-I156, R1-K155,R1-E154, R1-G153, R1-S152, R1-H151, R1-T150, R1-Q149, R1-C148, R1-G147,R1-I146, R1-L145, R1-T144, R1-R143, R1-N142, R1-Y141, R1-T140, R1-A139,R1-E138, R1-I137, R1-L136, R1-I135, R1-D134, R1-W133, R1-L132, R1-N131,R1-G130, R1-H129, R1-A128, R1-E127, R1-A126, R1-L125, R1-N124, R1-I123,R1-T122, R1-M121, R1-T120, R1-V119, R1-G118, R1-G117, R1-T116, R1-F115,R1-D114, R1-V113, R1-L112, R1-A111, R1-E110, R1-S109, R1-V108, R1-Q107,R1-G106, R1-S105, R1-Q104, R1-L103, R1-D102, R1-E101, R1-Y100, R1-S99,R1-G98, R1-S97, R1-L96, R1-K95, R1-A94, R1-Y93, R1-A92, R1-K91, R1-E90,R1-L89, R1-L88, R1-A87, R1-G86, R1-W85, R1-F84, R1-L83, R1-N82, R1-K81,R1-Y80, R1-T79, R1-S78, R1-S77, R1-V76, R1-F75, R1-V74, R1-L73, R1-Q72,R1-G71, R1-A70, R1-E69, R1-N68, R1-V67, R1-P66, R1-L65, R1-R64, R1-D63,R1-D62, R1-I61, R1-V60, R1-V59, R1-P58, R1-V57, R1-W56, R1-N55, R1-G54,R1-Y53, R1-H52, R1-W51, R1-F50, R1-W49, R1-F48, R1-R47, R1-F46, R1-I45,R1-G44, R1-A43, R1-Y42, R1-K41, R1-E40, R1-T39, R1-F38, R1-S37, R1-Q36,R1-N35, R1-L34, R1-P33, R1-V32, R1-V31, R1-R30, R1-S29, R1-L28, R1-I27,R1-D26, R1-Q25, R1-H24, R1-L23, R1-A22, R1-L21, R1-A20, R1-Q19, R1-L18,R1-A17, R1-A16, R1-L15, R1-F14, R1-W13, R1-C12, R1-D11, R1-G10, R1-I8,and/or R1-G7 of SEQ ID NO:92. Polynucleotide sequences encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these C-terminal CAN-12v1 active site domain deletionpolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein.

Alternatively, such polypeptides may comprise polypeptide sequencescorresponding, for example, to internal regions of the CAN-12v1 activesite domain (e.g., any combination of both N— and C-terminal CAN-12v1active site domain deletions) of SEQ ID NO:92. For example, internalregions could be defined by the equation NX to CX, where NX refers toany N-terminal amino acid position of the CAN-12v1 active site domain(SEQ ID NO:92), and where CX refers to any C-terminal amino acidposition of the CAN-12v1 active site domain (SEQ ID NO:92).Polynucleotides encoding these polypeptides are also provided. Thepresent invention also encompasses the use of these polypeptides as animmunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following CAN-12v1 active site domainamino acid substitutions are encompassed by the present invention:wherein R90 is substituted with either an A, C, D, E, F, G, H, I, K, L,M, N, P, Q, S, T, V, W, or Y; wherein L91 is substituted with either anA, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein D92is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W, or Y; wherein L93 is substituted with either an A, C, D, E,F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein C94 issubstituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein Q95 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein G96 is substitutedwith either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein 197 is substituted with either an A, C, D, E, F, G, H, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein V98 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; whereinG99 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y; wherein D100 is substituted with either an A, C, E,F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein C101 issubstituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein W102 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein F103 is substitutedwith either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein L104 is substituted with either an A, C, D, E, F, G, H, I, K,M, N, P, Q, R, S, T, V, W, or Y; wherein A105 is substituted with eithera C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinA106 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y; wherein L107 is substituted with either an A, C, D,E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein Q108 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S,T, V, W, or Y; wherein A109 is substituted with either a C, D, E, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L110 is substitutedwith either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY; wherein A111 is substituted with either a C, D, E, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein L112 is substituted with eitheran A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; whereinH113 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein Q114 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein D115 issubstituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein I116 is substituted with either an A, C, D, E, F,G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L117 is substitutedwith either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY; wherein S118 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, T, V, W, or Y; wherein R119 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; whereinV120 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, W, or Y; wherein V121 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein P122 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S,T, V, W, or Y; wherein L123 is substituted with either an A, C, D, E, F,G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein N124 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; wherein Q125 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, R, S, T, V, W, or Y; wherein S126 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; whereinF127 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein T128 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein E129 issubstituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein K130 is substituted with either an A, C, D, E, F,G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y131 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, orW; wherein A132 is substituted with either a C, D, E, F, G, H. I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein G133 is substituted with eitheran A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinI134 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein F135 is substituted with either an A, C,D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R136 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S,T, V, W, or Y; wherein F137 is substituted with either an A, C, D, E, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W138 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, orY; wherein F139 is substituted with either an A, C, D, E, G, H, I, K, L,M, N, P, Q, P, S, T, V, W, or Y; wherein W140 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; whereinH141 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein Y142 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein G143 issubstituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein N144 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein W145 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, orY; wherein V146 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, T, W, or Y; wherein P147 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; whereinV148 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, W, or Y; wherein V149 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein I150 issubstituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein D151 is substituted with either an A, C, E, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D152 is substitutedwith either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein R153 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, S, T, V, W, or Y; wherein L154 is substituted with eitheran A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; whereinP155 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,Q, R, S, T, V, W, or Y; wherein V156 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein N157 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S,T, V, W, or Y; wherein E158 is substituted with either an A, C, D, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A159 is substitutedwith either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein G160 is substituted with either an A, C, D, E, F, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein Q161 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; whereinL162 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P,Q, R, S, T, V, W, or Y; wherein V163 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein F164 issubstituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein V165 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein S166 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, orY; wherein S167 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, T, V, W, or Y; wherein T168 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; whereinY169 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, V, or W; wherein K170 is substituted with either an A, C,D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N171 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, P, S,T, V, W, or Y; wherein L172 is substituted with either an A, C, D, E, F,G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein F173 is substitutedwith either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein W174 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, T, V, or Y; wherein G175 is substituted with eitheran A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinA176 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y; wherein L177 is substituted with either an A, C, D,E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein L178 issubstituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S,T, V, W, or Y; wherein E179 is substituted with either an A, C, D, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K180 is substitutedwith either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, orY; wherein A181 is substituted with either a C, D, E, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein Y182 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; whereinA183 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y; wherein K184 is substituted with either an A, C, D,E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L185 issubstituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S,T, V, W, or Y; wherein S186 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein G187 is substitutedwith either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein S188 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, T, V, W, or Y; wherein Y189 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; whereinE190 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein D191 is substituted with either an A, C,E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L192 issubstituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S,T, V, W, or Y; wherein Q193 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein S194 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, orY; wherein G195 is substituted with either an A, C, D, E, F, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein Q196 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; whereinV197 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, W, or Y; wherein S198 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein E199 issubstituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein A200 is substituted with either a C, D, E, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L201 is substitutedwith either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY; wherein V202 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, T, W, or Y; wherein D203 is substituted withiheihran A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinF204 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein T205 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein G206 issubstituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein G207 is substituted with either an A, C, D, E, F,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V208 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, orY; wherein T209 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, V, W, or Y; wherein M210 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; whereinT211 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, V, W, or Y; wherein I212 is substituted with either an A, C,D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N213 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S,T, V, W, or Y; wherein L214 is substituted with either an A, C, D, E, F,G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein A215 is substitutedwith either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein E216 is substituted with either an A, C, D, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein A217 is substituted with eithera C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinH218 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein G219 is substituted with either an A, C,D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N220 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S,T, V, W, or Y; wherein L221 is substituted with either an A, C, D, E, F,G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein W222 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, orY; wherein D223 is substituted with either an A, C, E, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein I224 is substituted with eitheran A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinL225 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P,Q, R, S, T, V, W, or Y; wherein I226 is substituted with either an A, C,D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E227 issubstituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein A228 is substituted with either a C, D, E, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T229 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, orY; wherein Y230 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, T, V, or W; wherein N231 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; whereinR232 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, S, T, V, W, or Y; wherein T233 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein L234 issubstituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S,T, V, W, or Y; wherein I235 is substituted with either an A, C, D, E, F,G, H, K, L, M, N, P, Q, P, S, T, V, W, or Y; wherein G236 is substitutedwith either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein C237 is substituted with either an A, D, E, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein Q238 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; whereinT239 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, V, W, or Y; wherein H240 is substituted with either an A, C,D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S241 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,T, V, W, or Y; wherein G242 is substituted with either an A, C, D, E, F,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E243 is substitutedwith either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein K244 is substituted with either an A, C, D, E, F, G, H, I, L,M, N, P, Q, R, S, T, V, W, or Y; wherein I245 is substituted with eitheran A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinL246 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P,Q, R, S, T, V, W, or Y; wherein E247 is substituted with either an A, C,D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N248 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S,T, V, W, or Y; wherein G249 is substituted with either an A, C, D, E, F,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L250 is substitutedwith either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY; wherein V251 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, T, W, or Y; wherein E252 is substituted with eitheran A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinG253 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein H254 is substituted with either an A, C,D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A255 issubstituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein Y256 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein T257 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, orY; wherein L258 is substituted with either an A, C, D, E, F, G, H, I, K,M, N, P, Q, R, S, T, V, W, or Y; wherein T259 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; whereinG260 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein I261 is substituted with either an A, C,D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R262 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S,T, V, W, or Y; wherein K263 is substituted with either an A, C, D, E, F,G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V264 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, orY; wherein T265 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, V, W, or Y; wherein C266 is substituted with eitheran A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinK267 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P,Q, R, S, T, V, W, or Y; wherein H268 is substituted with either an A, C,D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R269 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S,T, V, W, or Y; wherein P270 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein E271 is substitutedwith either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein Y272 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, T, V, or W; wherein L273 is substituted with eitheran A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; whereinV274 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, W, or Y; wherein K275 is substituted with either an A, C,D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L276 issubstituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S,T, V, W, or Y; wherein R277 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein N278 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; wherein P279 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, Q, R, S, T, V, W, or Y; wherein W280 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; whereinG281 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein K282 is substituted with either an A, C,D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V283 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, W, or Y; wherein E284 is substituted with either an A, C, D, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W285 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, orY; wherein K286 is substituted with either an A, C, D, E, F, G, H, I, L,M, N, P, Q, R, S, T, V, W, or Y; wherein G287 is substituted with eitheran A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinD288 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein W289 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein S290 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,T, V, W, or Y; wherein D291 is substituted with either an A, C, E, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S292 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, orY; wherein S293 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, T, V, W, or Y; wherein S294 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; whereinK295 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P,Q, R, S, T, V, W, or Y; wherein W296 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein E297 issubstituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein L298 is substituted with either an A, C, D, E, F,G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein L299 is substitutedwith either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY; wherein S300 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, T, V, W, or Y; wherein P301 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; whereinK302 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P,Q, R, S, T, V, W, or Y; wherein E303 is substituted with either an A, C,D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K304 issubstituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S,T, V, W, or Y; wherein I305 is substituted with either an A, C, D, E, F,G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L306 is substitutedwith either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY; wherein L307 is substituted with either an A, C, D, E, F, G, H, I, K,M, N, P, Q, R, S, T, V, W, or Y; wherein L308 is substituted with eitheran A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; whereinR309 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, S, T, V, W, or Y; wherein K310 is substituted with either an A, C,D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D311 issubstituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein N312 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein D313 is substitutedwith either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein G314 is substituted with either an A, C, D, E, F, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein E315 is substituted with eitheran A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinF316 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein W317 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein M318 issubstituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S,T, V, W, or Y; wherein T319 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein L320 is substitutedwith either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY; wherein Q321 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, R, S, T, V, W, or Y; wherein D322 is substituted with eitheran A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinF323 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein K324 is substituted with either an A, C,D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T325 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, V, W, or Y; wherein H326 is substituted with either an A, C, D, E, F,G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F327 is substitutedwith either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein V328 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, T, W, or Y; wherein L329 is substituted with eitheran A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; whereinL330 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P,Q, P, S, T, V, W, or Y; and/or wherein V331 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y of SEQ IDNO:54, in addition to any combination thereof. The present inventionalso encompasses the use of these CAN-12v1 active site domain amino acidsubstituted polypeptides as immunogenic and/or antigenic epitopes asdescribed elsewhere herein.

In preferred embodiments, the following CAN-12v1 active site domainconservative amino acid substitutions are encompassed by the presentinvention: wherein R90 is substituted with either a K, or H; wherein L91is substituted with either an A, I, or V; wherein D92 is substitutedwith an E; wherein L93 is substituted with either an A, I, or V; whereinC94 is a C; wherein Q95 is substituted with a N; wherein G96 issubstituted with either an A, M, S, or T; wherein I97 is substitutedwith either an A, V, or L; wherein V98 is substituted with either an A,I, or L; wherein G99 is substituted with either an A, M, S, or T;wherein D100 is substituted with an E; wherein C101 is a C; wherein W102is either an F, or Y; wherein F103 is substituted with either a W, or Y;wherein L104 is substituted with either an A, I, or V; wherein A105 issubstituted with either a G, I, L, M, S, T, or V; wherein A106 issubstituted with either a G, I, L, M, S, T, or V; wherein L107 issubstituted with either an A, I, or V; wherein Q108 is substituted witha N; wherein A109 is substituted with either a G, I, L, M, S, T, or V;wherein L110 is substituted with either an A, I, or V; wherein A111 issubstituted with either a G, I, L, M, S, T, or V; wherein L112 issubstituted with either an A, I, or V; wherein H113 is substituted witheither a K, or R; wherein Q114 is substituted with a N; wherein D115 issubstituted with an E; wherein I116 is substituted with either an A, V,or L; wherein L117 is substituted with either an A, I, or V; whereinS118 is substituted with either an A, G, M, or T; wherein R119 issubstituted with either a K, or H; wherein V120 is substituted witheither an A, I, or L; wherein V121 is substituted with either an A, I,or L; wherein P122 is a P; wherein L123 is substituted with either an A,I, or V; wherein N124 is substituted with a Q; wherein Q125 issubstituted with a N; wherein S126 is substituted with either an A, G,M, or T; wherein F127 is substituted with either a W, or Y; wherein T128is substituted with either an A, G, M, or S; wherein E129 is substitutedwith a D; wherein K130 is substituted with either a R, or H; whereinY131 is either an F, or W; wherein A132 is substituted with either a G,I, L, M, S, T, or V; wherein G133 is substituted with either an A, M, S,or T; wherein I134 is substituted with either an A, V, or L; whereinF135 is substituted with either a W, or Y; wherein R136 is substitutedwith either a K, or H; wherein F137 is substituted with either a W, orY; wherein W138 is either an F, or Y; wherein F139 is substituted witheither a W, or Y; wherein W140 is either an F, or Y; wherein H141 issubstituted with either a K, or R; wherein Y142 is either an F, or W;wherein G143 is substituted with either an A, M, S, or T; wherein N144is substituted with a Q; wherein W145 is either an F, or Y; wherein V146is substituted with either an A, I, or L; wherein P147 is a P; whereinV148 is substituted with either an A, I, or L; wherein V149 issubstituted with either an A, I, or L; wherein I150 is substituted witheither an A, V, or L; wherein D151 is substituted with an E; whereinD152 is substituted with an E; wherein R153 is substituted with either aK, or H; wherein L154 is substituted with either an A, I, or V; whereinP155 is a P; wherein V156 is substituted with either an A, I, or L;wherein N157 is substituted with a Q; wherein E158 is substituted with aD; wherein A159 is substituted with either a G, I, L, M, S, T, or V;wherein G160 is substituted with either an A, M, S, or T; wherein Q161is substituted with a N; wherein L162 is substituted with either an A,I, or V; wherein V163 is substituted with either an A, I, or L; whereinF164 is substituted with either a W, or Y; wherein V165 is substitutedwith either an A, I, or L; wherein S166 is substituted with either an A,G, M, or T; wherein S167 is substituted with either an A, G, M, or T;wherein T168 is substituted with either an A, G, M, or S; wherein Y169is either an F, or W; wherein K170 is substituted with either a R, or H;wherein N171 is substituted with a Q; wherein L172 is substituted witheither an A, I, or V; wherein F173 is substituted with either a W, or Y;wherein W174 is either an F, or Y; wherein G175 is substituted witheither an A, M, S, or T; wherein A176 is substituted with either a G, I,L, M, S, T, or V; wherein L177 is substituted with either an A, I, or V;wherein L178 is substituted with either an A, I, or V; wherein E179 issubstituted with a D; wherein K180 is substituted with either a R, or H;wherein A181 is substituted with either a G, I, L, M, S, T, or V;wherein Y182 is either an F, or W; wherein A183 is substituted witheither a G, I, L, M, S, T, or V; wherein K184 is substituted with eithera R, or H; wherein L185 is substituted with either an A, I, or V;wherein S186 is substituted with either an A, G, M, or T; wherein G187is substituted with either an A, M, S, or T; wherein S188 is substitutedwith either an A, G, M, or T; wherein Y189 is either an F, or W; whereinE190 is substituted with a D; wherein D191 is substituted with an E;wherein L192 is substituted with either an A, I, or V; wherein Q193 issubstituted with a N; wherein S194 is substituted with either an A, G,M, or T; wherein G195 is substituted with either an A, M, S, or T;wherein Q196 is substituted with a N; wherein V197 is substituted witheither an A, I, or L; wherein S198 is substituted with either an A, G,M, or T; wherein E199 is substituted with a D; wherein A200 issubstituted with either a G, I, L, M, S, T, or V; wherein L201 issubstituted with either an A, I, or V; wherein V202 is substituted witheither an A, I, or L; wherein D203 is substituted with an E; whereinF204 is substituted with either a W, or Y; wherein T205 is substitutedwith either an A, G, M, or S; wherein G206 is substituted with either anA, M, S, or T; wherein G207 is substituted with either an A, M, S, or T;wherein V208 is substituted with either an A, I, or L; wherein T209 issubstituted with either an A, G, M, or S; wherein M210 is substitutedwith either an A, G, S, or T; wherein T211 is substituted with either anA, G, M, or S; wherein I212 is substituted with either an A, V, or L;wherein N213 is substituted with a Q; wherein L214 is substituted witheither an A, I, or V; wherein A215 is substituted with either a G, I, L,M, S, T, or V; wherein E216 is substituted with a D; wherein A217 issubstituted with either a G, I, L, M, S, T, or V; wherein H218 issubstituted with either a K, or R; wherein G219 is substituted witheither an A, M, S, or T; wherein N220 is substituted with a Q; whereinL221 is substituted with either an A, I, or V; wherein W222 is either anF, or Y; wherein D223 is substituted with an E; wherein I224 issubstituted with either an A, V, or L; wherein L225 is substituted witheither an A, I, or V; wherein I226 is substituted with either an A, V,or L; wherein E227 is substituted with a D; wherein A228 is substitutedwith either a G, I, L, M, S, T, or V; wherein T229 is substituted witheither an A, G, M, or S; wherein Y230 is either an F, or W; wherein N231is substituted with a Q; wherein R232 is substituted with either a K, orH; wherein T233 is substituted with either an A, G, M, or S; whereinL234 is substituted with either an A, I, or V; wherein I235 issubstituted with either an A, V, or L; wherein G236 is substituted witheither an A, M, S, or T; wherein C237 is a C; wherein Q238 issubstituted with a N; wherein T239 is substituted with either an A, G,M, or S; wherein H240 is substituted with either a K, or R; wherein S241is substituted with either an A, G, M, or T; wherein G242 is substitutedwith either an A, M, S, or T; wherein E243 is substituted with a D;wherein K244 is substituted with either a R, or H; wherein I245 issubstituted with either an A, V, or L; wherein L246 is substituted witheither an A, I, or V; wherein E247 is substituted with a D; wherein N248is substituted with a Q; wherein G249 is substituted with either an A,M, S, or T; wherein L250 is substituted with either an A, I, or V;wherein V251 is substituted with either an A, I, or L; wherein E252 issubstituted with a D; wherein G253 is substituted with either an A, M,S, or T; wherein H254 is substituted with either a K, or R; wherein A255is substituted with either a G, I, L, M, S, T, or V; wherein Y256 iseither an F, or W; wherein T257 is substituted with either an A, G, M,or S; wherein L258 is substituted with either an A, I, or V; whereinT259 is substituted with either an A, G, M, or S; wherein G260 issubstituted with either an A, M, S, or T; wherein I261 is substitutedwith either an A, V, or L; wherein R262 is substituted with either a K,or H; wherein K263 is substituted with either a R, or H; wherein V264 issubstituted with either an A, I, or L; wherein T265 is substituted witheither an A, G, M, or S; wherein C266 is a C; wherein K267 issubstituted with either a R, or H; wherein H268 is substituted witheither a K, or R; wherein R269 is substituted with either a K, or H;wherein P270 is a P; wherein E271 is substituted with a D; wherein Y272is either an F, or W; wherein L273 is substituted with either an A, I,or V; wherein V274 is substituted with either an A, I, or L; whereinK275 is substituted with either a R, or H; wherein L276 is substitutedwith either an A, I, or V; wherein R277 is substituted with either a K,or H; wherein N278 is substituted with a Q; wherein P279 is a P; whereinW280 is either an F, or Y; wherein G281 is substituted with either an A,M, S, or T; wherein K282 is substituted with either a R, or H; whereinV283 is substituted with either an A, I, or L; wherein E284 issubstituted with a D; wherein W285 is either an F, or Y; wherein K286 issubstituted with either a R, or H; wherein G287 is substituted witheither an A, M, S, or T; wherein D288 is substituted with an E; whereinW289 is either an F, or Y; wherein S290 is substituted with either an A,G, M, or T; wherein D291 is substituted with an E; wherein S292 issubstituted with either an A, G, M, or T; wherein S293 is substitutedwith either an A, G, M, or T; wherein S294 is substituted with either anA, G, M, or T; wherein K295 is substituted with either a R, or H;wherein W296 is either an F, or Y; wherein E297 is substituted with a D;wherein L298 is substituted with either an A, I, or V; wherein L299 issubstituted with either an A, I, or V; wherein S300 is substituted witheither an A, G, M, or T; wherein P301 is a P; wherein K302 issubstituted with either a R, or H; wherein E303 is substituted with a D;wherein K304 is substituted with either a R, or H; wherein I305 issubstituted with either an A, V, or L; wherein L306 is substituted witheither an A, I, or V; wherein L307 is substituted with either an A, I,or V; wherein L308 is substituted with either an A, I, or V; whereinR309 is substituted with either a K, or H; wherein K310 is substitutedwith either a R, or H; wherein D311 is substituted with an E; whereinN312 is substituted with a Q; wherein D313 is substituted with an E;wherein G314 is substituted with either an A, M, S, or T; wherein E315is substituted with a D; wherein F316 is substituted with either a W, orY; wherein W317 is either an F, or Y; wherein M318 is substituted witheither an A, G, S, or T; wherein T319 is substituted with either an A,G, M, or S; wherein L320 is substituted with either an A, I, or V;wherein Q321 is substituted with a N; wherein D322 is substituted withan E; wherein F323 is substituted with either a W, or Y; wherein K324 issubstituted with either a R, or H; wherein T325 is substituted witheither an A, G, M, or S; wherein H326 is substituted with either a K, orR; wherein F327 is substituted with either a W, or Y; wherein V328 issubstituted with either an A, I, or L; wherein L329 is substituted witheither an A, I, or V; wherein L330 is substituted with either an A, I,or V; and/or wherein V331 is substituted with either an A, I, or L ofSEQ ID NO:54 in addition to any combination thereof. Other suitablesubstitutions within the CAN-12v1 active site domain are encompassed bythe present invention and are referenced elsewhere herein. The presentinvention also encompasses the use of these CAN-12v1 active site domainconservative amino acid substituted polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

For purposes of the present invention, by “at least a portion of” ismeant all or any part of the CAN-12v1 active site domain correspondingto the analogous amino acids of the CAN-12 active site domain defined bythe structure coordinates according to Table IV (e.g., fragmentsthereof). More preferred are molecules comprising all or any parts ofthe CAN-12v1 active site domain, corresponding to the analogous aminoacids of the CAN-12 active site domain defined by the structurecoordinates according to Table IV, or a mutant or homologue of saidmolecule or molecular complex. By mutant or homologue of the molecule itis meant a molecule that has a root mean square deviation from thebackbone atoms of said CAN-12v1 amino acids of not more than 4.5Angstroms, and preferably not more than 3.5 Angstroms.

The term “root mean square deviation” means the square root of thearithmetic mean of the squares of the deviations from the mean. It is aterm that expresses the deviation or variation from a trend or object.For the purposes of the present invention, the “root mean squaredeviation” defines the variation in the backbone of a protein from therelevant portion of the backbone of the AR portion of the complex asdefined by the structure coordinates described herein.

A preferred embodiment is a machine-readable data storage medium that iscapable of displaying a graphical three-dimensional representation of amolecule or molecular complex that is defined by the structurecoordinates of all of the amino acids in Table IV ±a root mean squaredeviation from the backbone atoms of those amino acids of not more than4.0 ANG, preferably 3.0 ANG.

The structure coordinates of a CAN-12 homology model, including portionsthereof, is stored in a machine-readable storage medium. Such data maybe used for a variety of purposes, such as drug discovery.

Accordingly, in one embodiment of this invention is provided amachine-readable data storage medium comprising a data storage materialencoded with the structure coordinates set forth in Table IV.

One embodiment utilizes System 10 as disclosed in WO 98/11134, thedisclosure of which is incorporated herein by reference in its entirety.Briefly, one version of these embodiments comprises a computercomprising a central processing unit (“CPU”), a working memory which maybe, e.g, RAM (random-access memory) or “core” memory, mass storagememory (such as one or more disk drives or CD-ROM drives), one or morecathode-ray tube (“CRT”) display terminals, one or more keyboards, oneor more input lines, and one or more output lines, all of which areinterconnected by a conventional bidirectional system bus.

Input hardware, coupled to the computer by input lines, may beimplemented in a variety of ways. Machine-readable data of thisinvention may be inputted via the use of a modem or modems connected bya telephone line or dedicated data line. Alternatively or additionally,the input hardware may comprise CD-ROM drives or disk drives. Inconjunction with a display terminal, keyboard may also be used as aninput device.

Output hardware, coupled to the computer by output lines, may similarlybe implemented by conventional devices. By way of example, outputhardware may include a CRT display terminal for displaying a graphicalrepresentation of a region or domain of the present invention using aprogram such as QUANTA as described herein. Output hardware might alsoinclude a printer, so that hard copy output may be produced, or a diskdrive, to store system output for later use.

In operation, the CPU coordinates the use of the various input andoutput devices, coordinates data accesses from mass storage, andaccesses to and from the working memory, and determines the sequence ofdata processing steps. A number of programs may be used to process themachine-readable data of this invention. Such programs are discussed inreference to the computational methods of drug discovery as describedherein. Specific references to components of the hardware system areincluded as appropriate throughout the following description of the datastorage medium.

For the purpose of the present invention, any magnetic data storagemedium which can be encoded with machine-readable data would besufficient for carrying out the storage requirements of the system. Themedium could be a conventional floppy diskette or hard disk, having asuitable substrate, which may be conventional, and a suitable coating,which may be conventional, on one or both sides, containing magneticdomains whose polarity or orientation could be altered magnetically, forexample. The medium may also have an opening for receiving the spindleof a disk drive or other data storage device.

The magnetic domains of the coating of a medium may be polarized ororiented so as to encode in a manner which may be conventional, machinereadable data such as that described herein, for execution by a systemsuch as the system described herein.

Another example of a suitable storage medium which could also be encodedwith such machine-readable data, or set of instructions, which could becarried out by a system such as the system described herein, could be anoptically-readable data storage medium. The medium could be aconventional compact disk read only memory (CD-ROM) or a rewritablemedium such as a magneto-optical disk which is optically readable andmagneto-optically writable. The medium preferably has a suitablesubstrate, which may be conventional, and a suitable coating, which maybe conventional, usually of one side of substrate.

In the case of a CD-ROM, as is well known, the coating is reflective andis impressed with a plurality of pits to encode the machine-readabledata. The arrangement of pits is read by reflecting laser light off thesurface of the coating. A protective coating, which preferably issubstantially transparent, is provided on top of the reflective coating.

In the case of a magneto-optical disk, as is well known, the coating hasno pits, but has a plurality of magnetic domains whose polarity ororientation can be changed magnetically when heated above a certaintemperature, as by a laser. The orientation of the domains can be readby measuring the polarization of laser light reflected from the coating.The arrangement of the domains encodes the data as described above.

Thus, in accordance with the present invention, data capable ofdisplaying the three dimensional structure of the CAN-12 homology model,or portions thereof and their structurally similar homologues is storedin a machine-readable storage medium, which is capable of displaying agraphical three-dimensional representation of the structure. Such datamay be used for a variety of purposes, such as drug discovery.

For the first time, the present invention permits the use ofstructure-based or rational drug design techniques to design, select,and synthesize chemical entities that are capable of modulating thebiological function of CAN-12v1.

Accordingly, the present invention is also directed to the design ofsmall molecules which imitates the structure of the CAN-12v1 active sitedomain (SEQ ID NO:92), or a portion thereof, corresponding to theanalogous amino acids of the CAN-12 active site domain defined by thestructure provided in Table IV. Alternatively, the present invention isdirected to the design of small molecules which may bind to at leastpart of the CAN-12v1 active site domain (SEQ ID NO:92), or some portionthereof. For purposes of this invention, by CAN-12v1 active site domain,it is also meant to include mutants or homologues thereof. In apreferred embodiment, the mutants or homologues have at least 25%identity, more preferably 50% identity, more preferably 75% identity,and most preferably 90% identity to SEQ ID NO:92. In this context, theterm “small molecule” may be construed to mean any molecule describedknown in the art or described elsewhere herein, though may include, forexample, peptides, chemicals, carbohydrates, nucleic acids, PNAs, andany derivatives thereof.

The three-dimensional model structure of CAN-12v1, corresponding to thestructure coordinates of the analogous amino acids of the CAN-12polypeptide, will also provide methods for identifying modulators ofbiological function. Various methods or combination thereof can be usedto identify these compounds.

For example, test compounds can be modeled that fit spatially into theactive site domain in CAN-12v1 embodied by the sequence from about R90to about amino acid C94, from about amino acid G99 to about amino acidL104, from about amino acid Q196 to about amino acid S198, from aboutamino acid M210 to about amino acid I212, from about amino acid G236 toabout amino acid H240, from about amino acid E252 to about amino acidY256, from about amino acid N278 to about amino acid K282, from aboutamino acid V328 to about amino acid V331, or some portion thereof, ofSEQ ID NO:54 (corresponding to SEQ ID NO:92), in accordance with thestructural coordinates of the corresponding amino acids of the CAN-12polypeptide of Table IV.

Structure coordinates of the active site domain in CAN-12v1 defined bythe amino acids from about R90 to about amino acid C94, from about aminoacid G99 to about amino acid L104, from about amino acid Q196 to aboutamino acid S198, from about amino acid M210 to about amino acid I212,from about amino acid G236 to about amino acid H240, from about aminoacid E252 to about amino acid Y256, from about amino acid N278 to aboutamino acid K282, from about amino acid V328 to about amino acid V331 ofSEQ ID NO:54, can also be used to identify structural and chemicalfeatures. Identified structural or chemical features can then beemployed to design or select compounds as potential CAN-12v1 modulators.By structural and chemical features it is meant to include, but is notlimited to, van der Waals interactions, hydrogen bonding interactions,charge interaction, hydrophobic bonding interaction, and dipoleinteraction. Alternatively, or in conjunction with, thethree-dimensional structural model can be employed to design or selectcompounds as potential CAN-12v1 modulators. Compounds identified aspotential CAN-12v1 modulators can then be synthesized and screened in anassay characterized by binding of a test compound to the CAN-12v1, or incharacterizing the ability of CAN-12v1 to modulate a protease target inthe presence of a small molecule. Examples of assays useful in screeningof potential CAN-12v1 modulators include, but are not limited to,screening in silico, in vitro assays and high throughput assays.Finally, these methods may also involve modifying or replacing one ormore amino acids at amino acid positions, C101, H254, and/or N278 of SEQID NO:54 in accordance with the structure coordinates of thecorresponding amino acids of CAN-12 as provided in Table IV.

However, as will be understood by those of skill in the art upon thisdisclosure, other structure based design methods can be used. Variouscomputational structure based design methods have been disclosed in theart.

For example, a number of computer modeling systems are available inwhich the sequence of the CAN-12v1 and the CAN-12 structure (i.e.,atomic coordinates of CAN-12 and/or the atomic coordinates of the activesite domain as provided in Table IV, or the corresponding amino acids ofCAN-12v1 which are identical to the same region of CAN-12 and for whichthe same coordinates may be relied thereon) can be input. This computersystem then generates the structural details of one or more theseregions in which a potential CAN-12v1 modulator binds so thatcomplementary structural details of the potential modulators can bedetermined. Design in these modeling systems is generally based upon thecompound being capable of physically and structurally associating withCAN-12v1. In addition, the compound must be able to assume aconformation that allows it to associate with CAN-12v1. Some modelingsystems estimate the potential inhibitory or binding effect of apotential CAN-12v1 modulator prior to actual synthesis and testing.

Methods for screening chemical entities or fragments for their abilityto associate with a given protein target are also well known. Oftenthese methods begin by visual inspection of the binding site on thecomputer screen. Selected fragments or chemical entities are thenpositioned in the active site domain of CAN-12v1. Docking isaccomplished using software such as INSIGHTII, QUANTA and SYBYL,following by energy minimization and molecular dynamics with standardmolecular mechanic forcefields such as CHARS and AMBER. Examples ofcomputer programs which assist in the selection of chemical fragment orchemical entities useful in the present invention include, but are notlimited to, GRID (Goodford, 1985), AUTODOCK (Goodsell, 1990), and DOCK(Kuntz et al. 1982).

Upon selection of preferred chemical entities or fragments, theirrelationship to each other and CAN-12v1 can be visualized and thenassembled into a single potential modulator. Programs useful inassembling the individual chemical entities include, but are not limitedto CAVEAT (Bartlett et al. 1989) and 3D Database systems (Martin 1992).

Alternatively, compounds may be designed de novo using either an emptyactive site or optionally including some portion of a known inhibitor.Methods of this type of design include, but are not limited to LUDI(Bohm 1992) and LeapFrog (Tripos Associates, St. Louis Mo.).

In addition, CAN-12v1 is overall well suited to modem methods includingcombinatorial chemistry.

Programs such as DOCK (Kuntz et al. 1982) can be used with the atomiccoordinates from the homology model to identify potential ligands fromdatabases or virtual databases which potentially bind CAN-12 active sitedomain, and which may therefore be suitable candidates for synthesis andtesting.

Additionally, the three-dimensional homology model of CAN-12 will aid inthe design of mutants with altered biological activity for the CAN-12v1polypeptide.

The following are encompassed by the present invention: amachine-readable data storage medium, comprising a data storage materialencoded with machine readable data, wherein the data is defined by thestructure coordinates of the model CAN-12 according to Table IV or ahomologue of said model, wherein said homologue comprises backbone atomsthat have a root mean square deviation from the backbone atoms of thecomplex of not more than 4.5 Å, preferably not more than 4.0 Å, mostpreferably not more than 3.5 Å, and even more preferably not more than3.0 Å; and a machine-readable data storage medium, wherein said moleculeis defined by the set of structure coordinates of the model for CAN-12according to Table IV, or a homologue of said molecule, said homologuehaving a root mean square deviation from the backbone atoms of saidamino acids of not more than 4.5 Å, preferably not more than 4.0 Å, mostpreferably not more than 3.5 Å, and even more preferably not more than3.0 Å; a model comprising all or any part of the model defined bystructure coordinates of CAN-12 according to Table IV, or a mutant orhomologue of said molecule or molecular complex.

In a further embodiment, the following are encompassed by the presentinvention: a method for identifying a mutant of CAN-12v1 with alteredbiological properties, function, or reactivity, the method comprisingany combination of steps of: use of the model or a homologue of saidmodel according to Table IV, for the design of protein mutants withaltered biological function or properties which exhibit any combinationof therapeutic effects provided elsewhere herein; and use of the modelor a homologue of said model, for the design of a protein with mutationsin the active site domain comprised of the amino acids from about R90 toabout amino acid C94, from about amino acid G99 to about amino acidL104, from about amino acid Q196 to about amino acid S198, from aboutamino acid M210 to about amino acid I212, from about amino acid G236 toabout amino acid H240, from about amino acid E252 to about amino acidY256, from about amino acid N278 to about amino acid K282, from aboutamino acid V328 to about amino acid V331 of SEQ ID NO:54 according tothe corresponding amino acids of CAN-12 as provided in Table IV withaltered biological function or properties which exhibit any combinationof therapeutic effects provided elsewhere herein.

In further preferred embodiments, the following are encompassed by thepresent invention: a method for identifying modulators of CAN-12v1biological properties, function, or reactivity, the method comprisingany combination of steps of: modeling test compounds that overlayspatially into the active site domain defined by all or any portion ofresidues from about R90 to about amino acid C94, from about amino acidG99 to about amino acid L104, from about amino acid Q196 to about aminoacid S198, from about amino acid M210 to about amino acid I212, fromabout amino acid G236 to about amino acid H240, from about amino acidE252 to about amino acid Y256, from about amino acid N278 to about aminoacid K282, from about amino acid V328 to about amino acid V331 of SEQ IDNO:54 in reference to the corresponding amino acids of CAN-12 accordingto Table IV, or using a homologue or portion thereof.

The present invention encompasses using the structure coordinates as setforth herein to identify structural and chemical features of theCAN-12v1 polypeptide; employing identified structural or chemicalfeatures to design or select compounds as potential CAN-12v1 modulators;employing the three-dimensional structural model to design or selectcompounds as potential CAN-12v1 modulators; synthesizing the potentialCAN-12v1 modulators; screening the potential CAN-12v1 modulators in anassay characterized by binding of a protein to the CAN-12v1; selectingthe potential CAN-12v1 modulator from a database; designing the CAN-12v1modulator de novo; and/or designing said CAN-12v1 modulator from a knownmodulator activity.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO: 53 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 2081 ofSEQ ID NO:53, b is an integer between 15 to 2095, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ IDNO:53, and where b is greater than or equal to a+14.

In one embodiment, a CAN12.v1 polypeptide comprises a portion of theamino sequence depicted in FIGS. 8A-C. In another embodiment, a CAN12.v1polypeptide comprises at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 amino acids of the amino sequence depicted in FIGS.8A-C. In further embodiments, the CAN12.v1 polypeptide does not consistof the sequence ALLEKAYAKL (SEQ ID NO:141), and/or ALLEKAYAKLSGSYE. (SEQID NO:142).

Features of the Polypeptide Encoded by Gene No:3

The polypeptide of this gene provided as SEQ ID NO:56 (FIGS. 9A-C),encoded by the polynucleotide sequence according to SEQ ID NO:55 (FIGS.9A-C), and/or encoded by the polynucleotide contained within thedeposited clone, CAN-12v2, has significant homology at the nucleotideand amino acid level to a number of calpains, which include, forexample, the human CAN10 protein (type II diabetes linked) (hCAN10;Genbank Accession No: gi|[NP_(—)075574; SEQ ID NO:3); the human CAN5protein (hCAN5; Genbank Accession No: gi|[NP_(—)004046; SEQ ID NO:4);the large catalytic subunit of the human CALPAIN 1 protein (alsoreferred to as Calcium-Activated Neutral Proteinase, CANP, μ-TYPE)(hCAN1; Genbank Accession No: gi|12408656; SEQ ID NO:5); the largesubunit of the human calpain 3 protein (EC 3.4.22.17) (also referred toas CALPAIN L3, CALPAIN P94, Calcium-Activated Neutral Proteinase 3, CANP3; muscle-specific calcium-activated neutral protease 3 large subunit)(hCAN3; Genbank Accession No: gi|4557405; SEQ ID NO:6); the largecatalytic subunit of the mouse CALPAIN 1 protein (also referred to asCalcium-Activated Neutral Proteinase) (CANP) μ-TYPE ) (mCALPAIN1;Genbank Accession No: gi|088666; SEQ ID NO:7); the mouse CALPAIN LP82(mLP82; Genbank Accession No: gi|3661585; SEQ ID NO:8); the ratstomach-specific calcium-activated neutral protease large subunit, NCL2protein (rNCL2; Genbank Accession No: gi|NP-006606; SEQ ID NO:9); thehuman CAN11 protein (hCAN11; Genbank Accession No: gi|NP_(—)008989; SEQID NO:10); the human CAN2 protein (hCAN2; Genbank Accession No:gi|4502563; SEQ ID NO:11); and the human CAN9 protein (hCAN9; GenbankAccession No: gi|5729758; SEQ ID NO:12). An alignment of the CAN-12v2polypeptide with these proteins is provided in FIGS. 2A-E. Based uponsuch strong conservation, the inventors have ascribed the CAN-12v2polypeptide as having proteolytic activity, preferably calpain activity.

The CAN-12v2 polypeptide was determined to have 28.8% identity and 35.7%similarity with the human CAN10 protein (type II diabetes linked)(hCAN10; Genbank Accession No: gi|NP_(—)075574; SEQ ID NO:3); to have33.3% identity and 45.1% similarity with the human CAN5 protein (hCAN5;Genbank Accession No: gi|NP_(—)004046; SEQ ID NO:4); to have 38.3%identity and 46.6% similarity with the large catalytic subunit of thehuman CALPAIN 1 protein (also referred to as Calcium-Activated NeutralProteinase, CANP, μ-TYPE) (hCAN1; Genbank Accession No: gi|12408656; SEQID NO:5); to have 41.3% identity and 49.8% similarity with the largesubunit of the human calpain 3 protein (EC 3.4.22.17) (also referred toas CALPAIN L3, CALPAIN P94, Calcium-Activated Neutral Proteinase 3, CANP3; muscle-specific calcium-activated neutral protease 3 large subunit)(hCAN3; Genbank Accession No: gi|4557405; SEQ ID NO:6); to have 39.6%identity and 47.6% similarity with the large catalytic subunit of themouse CALPAIN 1 protein (also referred to as Calcium-Activated NeutralProteinase) (CANP) μ-TYPE ) (mCALPAIN1; Genbank Accession No: gi|088666;SEQ ID NO:7); to have 40.6% identity and 48.8% similarity with the mouseCALPAIN LP82 (mLP82; Genbank Accession No: gi|3661585; SEQ ID NO:8); tohave 36.3% identity and 44.9% similarity with the rat stomach-specificcalcium-activated neutral protease large subunit, NCL2 protein (rNCL2;Genbank Accession No: gi|NP_(—)006606; SEQ ID NO:9); to have 38.8%identity and 47.3% similarity with the human CAN11 protein (hCAN11;Genbank Accession No: gi|NP_(—)008989; SEQ ID NO:10); to have 37.9%identity and 47.3% similarity with the human CAN2 protein (hCAN2;Genbank Accession No: gi|4502563; SEQ ID NO:11); and to have 40.7%identity and 49.8% similarity with the human CAN9 protein (hCAN9;Genbank Accession No: gi|5729758; SEQ ID NO:12).

The human CAN10 protein (type II diabetes linked) (hCAN10; GenbankAccession No: gi|NP_(—)075574; SEQ ID NO:3)is a human calpain gene thatencodes a large calpain subunit. CAN10 is an atypical calpain in that itlacks the calmodulin-like calcium-binding domain and instead has adivergent C-terminal domain. CAN10 is similar in organization tocalpains 5 and 6 and is associated with type 2 or non-insulin-dependentdiabetes mellitus (NIDDM) and located within the NIDDM1 chromosomalregion (Nat. Genet. 26 (2), 163-175 (2000)).

The large subunit of the human calpain 3 protein (EC 3.4.22.17) (alsoreferred to as CALPAIN L3, CALPAIN P94, Calcium-Activated NeutralProteinase 3, CANP 3; muscle-specific calcium-activated neutral protease3 large subunit) (hCAN3; Genbank Accession No: gi|4557405; SEQ ID NO:6)is a muscle-specific member of the calpain large subunit family. Loss ofCAPN3 function has been associated with limb-girdle muscular dystrophiestype 2A (Cell 81 (1), 27-40 (1995)).

The human CAN9 protein (hCAN9; Genbank Accession No: gi|5729758; SEQ IDNO:12) is a calpain that is expressed predominantly in stomach and smallintestine and is thought to have specialized functions in the digestivetract, and be associated with gastric cancer.( Biol. Chem. 379 (2),175-183 (1998); and Jpn. J. Cancer Res. 91 (5), 459-463 (2000)).

As described above, the CAN-12v2 polypeptide was found to havesignificant sequence homology with calpains, particularly members of them-calpain family. A conserved peptide signature ofQx3(G,E)xC(Y,W)x2(S,T,A,G,C)(S,T,A,G,C,V) Qx{3}(G)xC(W)x{2}(A)(A)(referred to as a thiol (cysteine) protease active site domain) commonto most calpain family members is found in the protein sequence ofCAN-12v2 from amino acid 90 to amino acid 111 of SEQ ID NO:56 (FIGS.9A-C). Protein threading and molecular modeling of CAN-12v2 suggeststhat CAN-12v2 has a structural fold similar to representativem-calpains. Moreover, the structural and threading alignments of thepresent invention suggest that amino acids 101 (“C”), 254 (“H”), and 278(“N”) of SEQ ID NO:56 (FIGS. 9A-C) may represent the catalytic aminoacids within the active site domain. Thus, based upon the sequence andstructural homology to known calpains, particularly the presence of thethiol cysteine protease active site domain, the novel CAN-12v2 isbelieved to represent a novel human calpain.

In confirmation of the strong homology to known calpains, the CAN-12v2polypeptide was determined to have several conserved catalytic aminoacids at amino acid C101, H254, and N278 of SEQ ID NO:56 (FIGS. 9A-C).As discussed more particularly herein, calpains are a group ofstructurally diverse, high molecular weight (400 to 500 amino acids)proteins that have a catalytic cysteine amino acid and one or morecalcium binding domains. Despite the structural heterogeneity, calpainsshare some well defined structural-functional characteristics,particularly in their active site domains.

In preferred embodiments, the CAN-12v2 polypeptide of the presentinvention is directed to a polypeptide having structural similarity tocalpains.

Based upon the strong homology to members of the calpain family, theCAN-12v2 polypeptide is expected to share at least some biologicalactivity with calpains, preferably with m-calpain family members, andmore preferable to the large subunits of m-calpain family members, inaddition to other calpains and calpain subunits referenced herein and/orotherwise known in the art.

Expression profiling designed to measure the steady state mRNA levelsencoding the CAN-12 polypeptide showed predominately high expressionlevels in spinal cord tissue; significantly high expression in lymphnode and thymus, and to a lesser extent, in spleen tissue (See FIG. 4).

Expanded analysis of CAN-12 expression levels by TaqMan™ quantitativePCR (see FIG. 6) confirmed that the CAN-12 polypeptide is expressed inthe lymph gland. However, the TaqMan™ quantitative PCR determined thatthe CAN-12 polypeptide is primarily expressed in the eosophagus. Infact, with the exception of the lymph gland, the steady state mRNA levelof CAN-12 was approximately 2700 times higher in the esophagus than inall other tissues tested. These data suggest modulators of the CAN-12polynucleotides and polypeptides may be useful for the treatment,detection, and/or amelioration of the following, non-limiting diseasesand disorders associated with the eosphagus: dysphagia, cricoharyngealincoordination, esophageal carcinoma, esophageal webs, achalasia,symptomatic diffuse esophageal spasm, gastroesophageal reflux, and/orcorrosive esophagitis.

The polynucleotides encoding the CAN-12 polypeptide of the presentinvention were used to determine the chromosomal localization of thecalpain12 gene. Polynuclelotides corresponding to CAN-12 (SEQ ID NO:1)were shown to localize to chromosome 2, specifically 2p16-p21. Thecomparison of the chromosomal location of the calpain12 gene with thelocation of chromosomal regions which have been shown to be associatedwith specific diseases or conditions, e.g. by linkage analysis, can beindicative of diseases in which calpain12 may play a role.Interestingly, a whole-genome linkage scan in multiple sclerosisfamilies (Ebers et al. A full genome search in multiple sclerosis.Nature Genet. 13: 472-476, 1996.) identified 5 susceptibility loci onchromosomes 2, 3, 5, 11, and X. In particular, an association wasidentified with marker D2S119 on chromosome 2 and MS. The localizationof the D2S119 marker was further delimeated to 2p16-p21 based on aradiation hybrid linkage map retrieved from an online query at NCBI website: http:/www.ncbi.nlm.nih.gov/genome/sts/. Since the map of calpain12 and the susceptibility marker D2S119 overlaps, it is reasonable topostulate that calpain 12 may contribute to MS. Furthermore, thetranscription profile of calpain12 indicated a prominent expression inspinal cord, and implication of calpains in MS has been suggested(Shields D C et al. A putative mechanism of demyelination in multiplesclerosis by a proteolytic enzyme, calpain. Proc Natl Acad Sci USA.96:11486-91.1999).

The CAN-12v2 polynucleotides and polypeptides of the present invention,including agonists and/or fragments thereof, have uses that includemodulating cellular adhesion events, cellular proliferation, andinflammation, in various cells, tissues, and organisms, and particularlyin mammalian spinal cord tissue, lymph node, thymus, and spleen tissue,preferably human tissue. CAN-12v2 polynucleotides and polypeptides ofthe present invention, including agonists and/or fragments thereof, maybe useful in diagnosing, treating, prognosing, and/or preventing neural,immune, hematopoietic, and/or proliferative diseases or disorders.

The strong homology to human calpains, particularly m-calpains, combinedwith the predominate localized expression in esophagus tissue suggeststhe CAN-12 polynucleotides and polypeptides may be useful in treating,diagnosing, prognosing, and/or preventing gastrointestinal diseases,particularly esophageal diseases and/or disorders which include thefollowing non-limiting examples: aberrant transport of food bolus fromthe mouth to the stomach, aberrant prevention of retrograde flow ofgastrointestinal contents, aberrant esophageal peristaltic contractions,pyrosis, painful swallowing, reflux esophagitis, esophageal motilitydisorders, esophageal spasms, diffuse esophageal spasm, atypical chestpain, regurgitation, oropharyngeal paralysis, nasal regurgitation,dysphagia, cricopharyngeal bar, globus pharyngeus, achalasia, motordisorders of the esophageal smooth muscle, scleroderma esophagus,gastroesophageal reflux disease (GERD), esophagitis, Barrett'sesophagus, viral esophagitis, Herpes simplex virus mediated viralesophagitis, Varicella-zoster virus mediated viral esophagitis,Cytomegalovirus mediated viral esophagitis, bacterial esophagitis,Lactobacillus mediated bacterial esophagitis, Candida mediatedesophagitis, radiation esophagitis, corrosive esophagitis, pill-inducedesophagitis, esophagitis associated with mucocutaneous and systemicdiseases, diverticula, lower esophageal mucosal ring, lower esophagealmuscular ring, hiatal hernia, paraesophageal hernia, esophageal rupture,and/or Mallory-Weiss Syndrome.

Although calpains are typically associated primarily withneurogenerative conditions, their association in gastrointenstinaltissues has precedence. For example, the human CAN9 protein (hCAN9;Genbank Accession No: gi|5729758; SEQ ID NO:12) is predominatelyexpressed in the stomach and small intestine and is thought to beassociated with gastric cancers.

The strong homology to human calpains, particularly m-calpains, combinedwith the localized expression in spinal cord tissue suggests theCAN-12v2 polynucleotides and polypeptides may be useful in treating,diagnosing, prognosing, and/or preventing neural diseases,neurodegenerative disease states, behavioral disorders, or inflammatoryconditions. Representative uses are described in the “NeurologicalDiseases”, “Regeneration” and “Hyperproliferative Disorders” sectionsbelow, in the Examples, and elsewhere herein. Briefly, the uses include,but are not limited to the detection, treatment, and/or prevention ofAlzheimer's Disease, Parkinson's Disease, Huntington's Disease, TouretteSyndrome, meningitis, encephalitis, demyelinating diseases, peripheralneuropathies, neoplasia, trauma, congenital malformations, spinal cordinjuries, ischemia and infarction, aneurysms, hemorrhages,schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder,depression, panic disorder, learning disabilities, ALS, psychoses,autism, and altered behaviors, including disorders in feeding, sleeppatterns, balance, and perception. In addition, elevated expression ofthis gene product in regions of the brain indicates it plays a role innormal neural function. Potentially, this gene product is involved insynapse formation, neurotransmission, learning, cognition, homeostasis,or neuronal differentiation or survival. Furthermore, the protein mayalso be used to determine biological activity, to raise antibodies, astissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

Alternatively, the strong homology to human calpains, particularlym-calpains, combined with the localized expression in lymph node,thymus, and spleen tissue suggests the CAN-12v2 polynucleotides andpolypeptides may be useful in treating, diagnosing, prognosing,ameliorating, and/or preventing immune diseases and/or disorders.Representative uses are described in the “Immune Activity” and“Infectious Disease” sections below, and elsewhere herein. Briefly, thestrong expression in immune tissue indicates a role in regulating theproliferation; survival; differentiation; and/or activation ofhematopoietic cell lineages, including blood stem cells. The CAN-12v2polypeptide may also be useful as a preventative agent for immunologicaldisorders including arthritis, asthma, immunodeficiency diseases such asAIDS, leukemia, rheumatoid arthritis, granulomatous disease,inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia,psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity;immune reactions to transplanted organs and tissues, such ashost-versus-graft and graft-versus-host diseases, or autoimmunitydisorders, such as autoimmune infertility, lense tissue injury,demyelination, systemic lupus erythematosis, drug induced hemolyticanemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.Moreover, the protein may represent a secreted factor that influencesthe differentiation or behavior of other blood cells, or that recruitshematopoietic cells to sites of injury. Thus, this gene product may beuseful in the expansion of stem cells and committed progenitors ofvarious blood lineages, and in the differentiation and/or proliferationof various cell types.

Moreover, the protein would be useful in the detection, treatment,and/or prevention of a variety of vascular disorders and conditions,which include, but are not limited to miscrovascular disease, vascularleak syndrome, aneurysm, stroke, embolism, thrombosis, coronary arterydisease, arteriosclerosis, and/or atherosclerosis. Furthermore, theprotein may also be used to determine biological activity, raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

In addition, antagonists of the CAN-12v2 polynucleotides andpolypeptides may have uses that include diagnosing, treating,prognosing, and/or preventing diseases or disorders related to hypercalpain activity, which may include immune and/or proliferative diseasesor disorders, particularly thrombosis, embolism, and other blooddisorders. Therapeutic and/or pharmaceutical compositions comprising theCAN-12v2 polypeptides may be formulated to comprise heparin.

In addition, antagonists of the CAN-12v2 polynucleotides andpolypeptides may have uses that include diagnosing, treating,ameliorating, prognosing, and/or preventing diseases or disordersrelated to hyper calpain activity, which may include neuronalexcitotoxicity, ischemic stroke, hemoragic stroke, hypoxic stress,trauma, cell destruction, spinal cord injury following trauma,degeneration of vulnerable hippocampal neurons after ischemia,reovirus-induced apoptosis, viral-induced induced myocarditis, acute andchronic inflammation, cataract formation, multiple sclerosis,demylenating disorders, acoustic trauma, hearing loss caused by noise,neuronal damage, cardiac ischemic damage, and/or hepatocyte necrosisduring and following anoxia.

CAN-12v2 polynucleotides and polypeptides, including fragments and/orantagonsists thereof, may have uses which include modulatingdevelopment, differentiation, cellular transformation in response tocell signaling, cell-cell and/or cell-extracellular matrix interactions,clustering of the integrin receptor aIIb3, modulating in long termpotentiation (memory), modulating neurite outgrowth, modulating corticallamination activation of protein kinases and phosphatases, remodelingand disassembling the cytoskeleton, cell cycle modulation, in addition,to ameliorating, preventing, and/or treating limb-girdle musculardystrophy (LGMD), insulin resistance in diabetics, Alzheimer's disease,Multiple sclerosis, Huntington's disease, Parkinson's disease andamyotrophy.

Moreover, CAN-12v2 polynucleotides and polypeptides, including fragmentsand agonists thereof, may have uses which include treating, diagnosing,prognosing, and/or preventing hyperproliferative disorders, particularlyof the neural and immune systems. Such disorders may include, forexample, cancers, and metastatic conditions.

CAN-12v2 polynucleotides and polypeptides, including fragments and/orantagonsists thereof, may have uses which include identification ofmodulators of CAN-12v2 function including antibodies (for detection orneutralization), naturally-occurring modulators and small moleculemodulators. Antibodies to domains (including CAN-12v2 epitopes providedherein) of the CAN-12v2 protein could be used as diagnostic agents ofinflammatory conditions in patients, are useful in monitoring theactivation and presence of cognate proteases, and can be used as abiomarker for the protease involvement in disease states and in theevaluation of inhibitors of the cognate protease in vivo.

CAN-12v2 polypeptides and polynucleotides are useful for diagnosingdiseases related to over or under expression of CAN-12v2 proteins byidentifying mutations in the CAN-12v2 gene using CAN-12v2 probes, ordetermining CAN-12v2 protein or mRNA expression levels. CAN-12v2polypeptides are also useful for screening for compounds, which affectactivity of the protein. Diseases that can be treated with CAN-12v2include, the following, non-limiting examples: neuro-regeneration,neuropathic pain, obesity, anorexia, HIV infections, cancers, bulimia,asthma, Parkinson's disease, acute heart failure, hypotension,hypertension, osteoporosis, angina pectoris, myocardial infarction,psychotic, immune, metabolic, cardiovascular, and neurologicaldisorders.

The predominate expression in neural tissues, combined with thesignificant expression in a number of other tissues, suggests theCAN-12v2 polynucleotide and polypeptide of the present invention may beinvolved in modulating nerve invasion, innervation, nerve maintenance,and potentially myeline sheath maintenance and integrity.

The CAN-12v2 polynucleotides and polypeptides, including fragments andantagonists thereof, may have uses which include detecting, diagnosing,treating, ameliorating, and/or preventing diseases and disorders of theneural system, particularly Alzheimer's disease, either directly orindirectly, in addition to other neural disorders known in the art orprovided in the “Neurological Diseases” section herein, such asmodulating nerve invasion, innervation, nerve maintenance, potentiallymyelin sheath maintenance and integrity, encephalomyelitis, autoimmuneencephalomyelitis, human T cell leukemia virus type I(HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP),and neuro-inflammatory diseases.

Molecular genetic manipulation of the structure of the active sitedomain, particularly the predicted catalytic amino acids, and of otherfunctional domains in the calpain family (e.g., active site domainbinding pocket) enables the production of calpains with tailor-madeactivities. Thus, the CAN-12v2 polypeptides, and fragments thereof, aswell as any homologous product resulting from genetic manipulation ofthe structure, are useful for NMR-based design of modulators of CAN-12v2biological activity, and calpains, in general.

CAN-12v2 polypeptides and polynucleotides have additional uses whichinclude diagnosing diseases related to the over and/or under expressionof CAN-12v2 by identifying mutations in the CAN-12v2 gene by usingCAN-12v2 sequences as probes or by determining CAN-12v2 protein or mRNAexpression levels. CAN-12v2 polypeptides may be useful for screeningcompounds that affect the activity of the protein. CAN-12v2 peptides canalso be used for the generation of specific antibodies and as bait inyeast two hybrid screens to find proteins the specifically interact withCAN-12v2 (described elsewhere herein).

The CAN-12v2 polynucleotides and polypeptides, including fragments andagonists thereof, may have uses which include detecting, diagnosing,treating, ameliorating, and/or preventing metabolic diseases anddisorders, such as diabetes. Moreover, expressed human CAN-12v2 may beuseful in the detection of patients susceptible to diabetes. Alsoparadigms that would simulate intracellular CAN-12v2 activity would beuseful in treating diabetes.

The CAN-12v2 polynucleotides and polypeptides, including fragmentsthereof, may have uses which include identifying inhibitors ofintracellular calpain inhibitors (calpastatins) leading to an effectiveincrease in calpain activity.

Various approaches to detect alterations or allelic variants at thegenomic or MRNA level of CAN-12v2, could be used as a diagnostic foridentifying MS patients, or individuals susceptible to have MS. It islikely that the CAN-12v2 gene comprises polymorphic sites (i.e. SNPs),with specific alleles which may be associated with MS or otherneurodegenerative disorders, or associated with an increased likelihoodof developing these diseases. Therefore, the invention provides theCAN-12v2 sequence that can be used to design specific primers for theidentification of polymorphisms or mutations in CAN-12v2 of patientsaffected with MS. The presence of a specific allele variant, such as aSNP allele or SNPs haplotype that renders the subject carrying it moresusceptible to develop MS or other related diseases could be identified(e.g. a variant in the CAN-12v2 promoter region that increasedtranscript levels of CAN-12v2, or mutations in the coding sequence thatincreased the stability or half-life of the CAN-12v2 protein). Othermethods such as Northern-blot analysis could be performed to measuretranscript levels using a CAN-12v2 cDNA probe derived from the sequenceof the invention.

Although it is believed the encoded polypeptide may share at least somebiological activities with human calpains (particularly m-calpains), anumber of methods of determining the exact biological function of thisclone are either known in the art or are described elsewhere herein.Briefly, the function of this clone may be determined by applyingmicroarray methodology. Nucleic acids corresponding to the CAN-12v2polynucleotides, in addition to, other clones of the present invention,may be arrayed on microchips for expression profiling. Depending onwhich polynucleotide probe is used to hybridize to the slides, a changein expression of a specific gene may provide additional insight into thefunction of this gene based upon the conditions being studied. Forexample, an observed increase or decrease in expression levels when thepolynucleotide probe used comes from diseased neural tissue, as comparedto, normal tissue might indicate a function in modulating neuralfunction, for example. In the case of CAN-12v2, spinal cord, lymph node,thymus, and/or spleen tissue should be used to extract RNA to preparethe probe.

In addition, the function of the protein may be assessed by applyingquantitative PCR methodology, for example. Real time quantitative PCRwould provide the capability of following the expression of the CAN-12v2gene throughout development, for example. Quantitative PCR methodologyrequires only a nominal amount of tissue from each developmentallyimportant step is needed to perform such experiments. Therefore, theapplication of quantitative PCR methodology to refining the biologicalfunction of this polypeptide is encompassed by the present invention. Inthe case of CAN-12v2, a disease correlation related to CAN-12v2 may bemade by comparing the mRNA expression level of CAN-12v2 in normaltissue, as compared to diseased tissue (particularly diseased tissueisolated from the following: esophagus, spinal cord, lymph node, thymus,and/or spleen tissue). Significantly higher or lower levels of CAN-12v2expression in the diseased tissue may suggest CAN-12v2 plays a role indisease progression, and antagonists against CAN-12v2 polypeptides wouldbe useful therapeutically in treating, preventing, and/or amelioratingthe disease. Alternatively, significantly higher or lower levels ofCAN-12v2 expression in the diseased tissue may suggest CAN-12v2 plays adefensive role against disease progression, and agonists of CAN-12v2polypeptides may be useful therapeutically in treating, preventing,and/or ameliorating the disease. Also encompassed by the presentinvention are quantitative PCR probes corresponding to thepolynucleotide sequence provided as SEQ ID NO:55 (FIGS. 9A-C).

The function of the protein may also be assessed through complementationassays in yeast. For example, in the case of the CAN-12v2, transformingyeast deficient in calpain activity, particularly m-calpain activity,and assessing their ability to grow would provide convincing evidencethe CAN-12v2 polypeptide has calpain activity, and possibly m-calpainactivity. Additional assay conditions and methods that may be used inassessing the function of the polynucleotides and polypeptides of thepresent invention are known in the art, some of which are disclosedelsewhere herein.

Alternatively, the biological function of the encoded polypeptide may bedetermined by disrupting a homologue of this polypeptide in Mice and/orrats and observing the resulting phenotype. Such knock-out experimentsare known in the art, some of which are disclosed elsewhere herein.

Moreover, the biological function of this polypeptide may be determinedby the application of antisense and/or sense methodology and theresulting generation of transgenic mice and/or rats. Expressing aparticular gene in either sense or antisense orientation in a transgenicmouse or rat could lead to respectively higher or lower expressionlevels of that particular gene. Altering the endogenous expressionlevels of a gene can lead to the observation of a particular phenotypethat can then be used to derive indications on the function of the gene.The gene can be either over-expressed or under expressed in every cellof the organism at all times using a strong ubiquitous promoter, or itcould be expressed in one or more discrete parts of the organism using awell characterized tissue-specific promoter (e.g., an esophagus, spinalcord, lymph node, thymus, or spleen specific promoter), or it can beexpressed at a specified time of development using an inducible and/or adevelopmentally regulated promoter.

In the case of CAN-12v2 transgenic mice or rats, if no phenotype isapparent in normal growth conditions, observing the organism underdiseased conditions (neural, immune, hematopoietic diseases ordisorders, cancers, etc.) may lead to understanding the function of thegene. Therefore, the application of antisense and/or sense methodologyto the creation of transgenic mice or rats to refine the biologicalfunction of the polypeptide is encompassed by the present invention.

In preferred embodiments, the following N-terminal CAN-12v2 deletionpolypeptides are encompassed by the present invention: M1-L697, S2-L697,L3-L697, W4-L697, P5-L697, P6-L697, F7-L697, R8-L697, C9-L697, R10-L697,W11-L697, K12-L697, L13-L697, A14-L697, P15-L697, R16-L697, Y17-L697,S18-L697, R19-L697, R20-L697, A21-L697, S22-L697, P23-L697, Q24-L697,Q25-L697, P26-L697, Q27-L697, Q28-L697, D29-L697, F30-L697, E31-L697,A32-L697, L33-L697, L34-L697, A35-L697, E36-L697, C37-L697, L38-L697,R39-L697, N40-L697, G41-L697, C42-L697, L43-L697, F44-L697, E45-L697,D46-L697, T47-L697, S48-L697, F49-L697, P50-L697, A51-L697, T52-L697,L53-L697, S54-L697, S55-L697, I56-L697, G57-L697, S58-L697, G59-L697,S60-L697, L61-L697, L62-L697, Q63-L697, K64-L697, L65-L697, P66-L697,P67-L697, R68-L697, L69-L697, Q70-L697, W71-L697, K72-L697, R73-L697,P74-L697, P75-L697, E76-L697, L77-L697, H78-L697, S79-L697, N80-L697,P81-L697, Q82-L697, F83-L697, Y84-L697, F85-L697, A86-L697, K87-L697,A88-L697, K89-L697, R90-L697, L91-L697, D92-L697, L93-L697, C94-L697,Q95-L697, G96-L697, I97-L697, V98-L697, G99-L697, D100-L697, C101-L697,W102-L697, F103-L697, L104-L697, A105-L697, A106-L697, L107-L697,Q108-L697, A109-L697, L110-L697, A111-L697, L112-L697, H113-L697,Q114-L697, D115-L697, I116-L697, L117-L697, S118-L697, R119-L697,V120-L697, V121-L697, P122-L697, L123-L697, N124-L697, Q125-L697,S126-L697, F127-L697, T128-L697, E129-L697, K130-L697, Y131-L697,A132-L697, G133-L697, I134-L697, F135-L697, R136-L697, F137-L697,W138-L697, F139-L697, W140-L697, H141-L697, Y142-L697, G143-L697,N144-L697, W145-L697, V146-L697, P147-L697, V148-L697, V149-L697,I150-L697, D151-L697, D152-L697, R153-L697, L154-L697, P155-L697,V156-L697, N157-L697, E158-L697, A159-L697, G160-L697, Q161-L697,L162-L697, V163-L697, F164-L697, V165-L697, S166-L697, S167-L697,T168-L697, Y169-L697, K170-L697, N171-L697, L172-L697, F173-L697,W174-L697, G175-L697, A176-L697, L177-L697, L178-L697, E179-L697,K180-L697, A181-L697, Y182-L697, A183-L697, K184-L697, L185-L697,S186-L697, G187-L697, S188-L697, Y189-L697, E190-L697, D191-L697,L192-L697, Q193-L697, S194-L697, G195-L697, Q196-L697, V197-L697,S198-L697, E199-L697, A200-L697, L201-L697, V202-L697, D203-L697,F204-L697, T205-L697, G206-L697, G207-L697, V208-L697, T209-L697,M210-L697, T211-L697, I212-L697, N213-L697, L214-L697, A215-L697,E216-L697, A217-L697, H218-L697, G219-L697, N220-L697, L221-L697,W222-L697, D223-L697, I224-L697, L225-L697, I226-L697, E227-L697,A228-L697, T229-L697, Y230-L697, N231-L697, R232-L697, T233-L697,L234-L697, I235-L697, G236-L697, C237-L697, Q238-L697, T239-L697,H240-L697, S241-L697, G242-L697, E243-L697, K244-L697, I245-L697,L246-L697, E247-L697, N248-L697, G249-L697, L250-L697, V251-L697,E252-L697, G253-L697, H254-L697, A255-L697, Y256-L697, T257-L697,L258-L697, T259-L697, G260-L697, I261-L697, R262-L697, K263-L697,V264-L697, T265-L697, C266-L697, K267-L697, H268-L697, R269-L697,P270-L697, E271-L697, Y272-L697, L273-L697, V274-L697, K275-L697,L276-L697, R277-L697, N278-L697, P279-L697, W280-L697, G281-L697,K282-L697, V283-L697, E284-L697, W285-L697, K286-L697, G287-L697,D288-L697, W289-L697, S290-L697, D291 -L697, S292-L697, S293-L697,S294-L697, K295-L697, W296-L697, E297-L697, L298-L697, L299-L697,S300-L697, P301-L697, K302-L697, E303-L697, K304-L697, I305-L697,L306-L697, L307-L697, L308-L697, R309-L697, K310-L697, D311-L697,N312-L697, D313-L697, G314-L697, E315-L697, F316-L697, W317-L697,M318-L697, T319-L697, L320-L697, Q321-L697, D322-L697, F323-L697,K324-L697, T325-L697, H326-L697, F327-L697, V328-L697, L329-L697,L330-L697, V331-L697, I332-L697, C333-L697, K334-L697, L335-L697,T336-L697, P337-L697, G338-L697, L339-L697, L340-L697, S341-L697,Q342-L697, E343-L697, A344-L697, A345-L697, Q346-L697, K347-L697,W348-L697, T349-L697, Y350-L697, T351-L697, M352-L697, R353-L697,E354-L697, G355-L697, R356-L697, W357-L697, E358-L697, K359-L697,R360-L697, S361-L697, T362-L697, A363-L697, G364-L697, G365-L697,Q366-L697, R367-L697, Q368-L697, L369-L697, L370-L697, Q371-L697,D372-L697, T373-L697, F374-L697, W375-L697, K376-L697, N377-L697,P378-L697, Q379-L697, F380-L697, L381-L697, L382-L697, S383-L697,V384-L697, W385-L697, R386-L697, P387-L697, E388-L697, E389-L697,G390-L697, R391-L697, R392-L697, S393-L697, L394-L697, R395-L697,P396-L697, C397-L697, S398-L697, V399-L697, L400-L697, V401-L697,S402-L697, L403-L697, L404-L697, Q405-L697, K406-L697, P407-L697,R408-L697, H409-L697, R410-L697, C411-L697, R412-L697, K413-L697,R414-L697, K415-L697, P416-L697, L417-L697, L418-L697, A419-L697,I420-L697, G421-L697, F422-L697, Y423-L697, L424-L697, Y425-L697,R426-L697, M427-L697, N428-L697, K429-L697, Y430-L697, H431-L697,D432-L697, D433-L697, Q434-L697, R435-L697, R436-697, L437-L697,P438-L697, P439-L697, E440-L697, F441-L697, F442-L697, Q443-L697,R444-L697, N445-L697, T446-L697, P447-L697, L448-L697, S449-L697,Q440-L697, P451-L697, D452-L697, R453-L697, F454-L697, L455-L697,K456-L697, E457-L697, K458-L697, E459-L697, V460-L697, S461-L697,Q462-L697, E463-L697, L464-L697, C465-L697, L466-L697, E467-L697,P468-L697, G469-L697, T470-L697, Y471-L697, L472-L697, I473-L697,V474-L697, P475-L697, C476-L697, I477-L697, L478-L697, E479-L697,A480-L697, H481-L697, Q482-L697, K483-L697, S484-L697, E485-L697,F486-L697, V487-L697, L488-L697, R489-L697, V490-L697, F491-L697,S492-L697, R493-L697, K494-L697, H495-L697, I496-L697, F497-L697,Y498-L697, E499-L697, I500-L697, G501-L697, S502-L697, N503-L697,S504-L697, G505-L697, V506-L697, V507-L697, F508-L697, S509-L697,K510-L697, E511-L697, I512-L697, E513-L697, D514-L697, Q515-L697,N516-L697, E517-L697, R518-L697, Q519-L697, D520-L697, E521-L697,F522-L697, F523-L697, T524-L697, K525-L697, F526-L697, F527-L697,E528-L697, K529-L697, H530-L697, P531-L697, E532-L697, I533-L697,N534-L697, A535-L697, V536-L697, Q537-L697, L538-L697, Q539-L697,N540-L697, L541-L697, L542-L697, N543-L697, Q544-L697, and/or M545-L697of SEQ ID NO:56. Polynucleotide sequences encoding these polypeptidesare also provided. The present invention also encompasses the use ofthese N-terminal CAN-12v2 deletion polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal CAN-12v2 deletionpolypeptides are encompassed by the present invention: M1-L697, M1-L696,M1-T695, M1-T694, M1-N693, M1-F692, M1-I691, M1-L690, M1-Y689, M1-S688,M1-T687, M1-S686, M1-H685, M1-A684, M1-V683, M1-G682, M1-G681, M1-D680,M1-I679, M1-N678, M1-A677, M1-L676, M1-I675, M1-V674, M1-P673, M1-V672,M1-I671, M1-M670, M1-F669, M1-F668, M1-T667, M1-P666, M1-T665, M1-S664,M1-Q663, M1-L662, M1-D661, M1-V660, M1-D659, M1-K658, M1-L657, M1-T656,M1-V655, M1-S654, M1-R653, M1-I652, M1-L651, M1-T650, M1-V649, M1-E648,M1-A647, M1-H646, M1-W645, M1-V644, M1-D643, M1-G642, M1-R641, M1-R640,M1-Q639, M1-R638, M1-I637, M1-L636, M1-T635, M1-C634, M1-G633, M1-A632,M1-R631, M1-T630, M1-H629, M1-G628, M1-C627, M1-S626, M1-W625, M1-S624,M1-K623, M1-R622, M1-H621, M1-R620, M1-G619, M1-A618, M1-E617, M1-R616,M1-M615, M1-A614, M1-A613, M1-H612, M1-L611, M1-Q610, M1-E609, M1-W608,M1-N607, M1-L606, M1-Y605, M1-G604, M1-S603, M1-G602, M1-R601, M1-D600,M1-Q599, M1-K598, M1-H597, M1-F596, M1-V595, M1-K594, M1-Q593, M1-S592,M1-L591, M1-K590, M1-L589, M1-Q588, M1-K587, M1-W586, M1-L585, M1-D584,M1-R583, M1-F582, M1-E581, M1-Q580, M1-I579, M1-S578, M1-M577, M1-T576,M1-G575, M1-S574, M1-A573, M1-N572, M1-L571, M1-D570, M1-L569, M1-L568,M1-A567, M1-L566, M1-I565, M1-G564, M1-Q563, M1-C562, M1-A561, M1-E560,M1-L559, M1-S558, M1-F557, M1-F556, M1-P555, M1-Q554, M1-R553, M1-S552,M1-G551, M1-L550, M1-S549, M1-S548, M1-W547, M1-T546, M1-M545, M1-Q544,M1-N543, M1-L542, M1-L541, M1-N540, M1-Q539, M1-L538, M1-Q537, M1-V536,M1-A535, M1-N534, M1-I533, M1-E532, M1-P531, M1-H530, M1-K529, M1-E528,M1-F527, M1-F526, M1-K525, M1-T524, M1-F523, M1-F522, M1-E521, M1-D520,M1-Q519, M1-R518, M1-E517, M1-N516, M1-Q515, M1-D514, M1-E513, M1-I512,M1-E511, M1-K510, M1-S509, M1-F508, M1-V507, M1-V506, M1-G505, M1-S504,M1-N503, M1-S502, M1-G501, M1-I500, M1-E499, M1-Y498, M1-F497, M1-I496,M1-H495, M1-K494, M1-R493, M1-S492, M1-F491, M1-V490, M1-R489, M1-L488,M1-V487, M1-F486, M1-E485, M1-S484, M1-K483, M1-Q482, M1-H481, M1-A480,M1-E479, M1-L478, M1-I477, M1-C476, M1-P475, M1-V474, M1-I473, M1-L472,M1-Y471, M1-T470, M1-G469, M1-P468, M1-E467, M1-L466, M1-C465, M1-L464,M1-E463, M1-Q462, M1-S461, M1-V460, M1-E459, M1-K458, M1-E457, M1-K456,M1-L455, M1-F454, M1-R453, M1-D452, M1-P451, M1-Q450, M1-S449, M1-L448,M1-P447, M1-T446, M1-N445, M1-R444, M1-Q443, M1-F442, M1-F441, M1-E440,M1-P439, M1-P438, M1-L437, M1-R436, M1-R435, M1-Q434, M1-D433, M1-D432,M1-H431, M1-Y430, M1-K429, M1-N428, and/or M1-M427 of SEQ ID NO:56.Polynucleotide sequences encoding these polypeptides are also provided.The present invention also encompasses the use of these C-terminalCAN-12v2 deletion polypeptides as immunogenic and/or antigenic epitopesas described elsewhere herein.

Alternatively, preferred polypeptides of the present invention maycomprise polypeptide sequences corresponding to, for example, internalregions of the CAN-12v2 polypeptide (e.g., any combination of both N—and C-terminal CAN-12v2 polypeptide deletions) of SEQ ID NO:56. Forexample, internal regions could be defined by the equation: amino acidNX to amino acid CX, wherein NX refers to any N-terminal deletionpolypeptide amino acid of CAN-12v2 (SEQ ID NO:56), and where CX refersto any C-terminal deletion polypeptide amino acid of CAN-12v2 (SEQ IDNO:56). Polynucleotides encoding these polypeptides are also provided.The present invention also encompasses the use of these polypeptides asan immunogenic and/or antigenic epitope as described elsewhere herein.

The present invention also encompasses immunogenic and/or antigenicepitopes of the CAN-12v2 polypeptide.

The CAN-12v2 polypeptides of the present invention were determined tocomprise several phosphorylation sites based upon the Motif algorithm(Genetics Computer Group, Inc.). The phosphorylation of such sites mayregulate some biological activity of the CAN-12v2 polypeptide. Forexample, phosphorylation at specific sites may be involved in regulatingthe proteins ability to associate or bind to other molecules (e.g.,proteins, ligands, substrates, DNA, etc.). In the present case,phosphorylation may modulate the ability of the CAN-12v2 polypeptide toassociate with other polypeptides, particularly the serine proteasesubstrate for CAN-12v2, or its ability to modulate serine proteasefunction.

The CAN-12v2 polypeptide was predicted to comprise eleven PKCphosphorylation sites using the Motif algorithm (Genetics ComputerGroup, Inc.). In vivo, protein kinase C exhibits a preference for thephosphorylation of serine or threonine residues. The PKC phosphorylationsites have the following consensus pattern: [ST]-x-[RK], where S or Trepresents the site of phosphorylation and ‘x’ an intervening amino acidresidue. Additional information regarding PKC phosphorylation sites canbe found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem.161:177-184(1986), and Kishimoto A., Nishiyama K., Nakanishi H.,Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem.260:12492-12499(1985); which are hereby incorporated by referenceherein.

In preferred embodiments, the following PKC phosphorylation sitepolypeptides are encompassed by the present invention: LAPRYSRRASPQQ(SEQ ID NO:76), LNQSFTEKYAGIF (SEQ ID NO:77), VFVSSTYKNLFWG (SEQ IDNO:78), GIRKVTCKHRPEY (SEQ ID NO:79), DWSDSSSKWELLS (SEQ ID NO:80),KWELLSPKEKILL (SEQ ID NO:81), QKWTYTMREGRWE (SEQ ID NO:82),EEGRRSLRPCSVL (SEQ ID NO:83), VLRVFSRKHIFYE (SEQ ID NO:84),KQLKLSQKVFHKQ (SEQ ID NO:85), and/or LIRSVTLKDVDLQ (SEQ ID NO:86).Polynucleotides encoding these polypeptides are also provided. Thepresent invention also encompasses the use of the CAN-12v2 PKCphosphorylation site polypeptides as immunogenic and/or antigenicepitopes as described elsewhere herein.

The CAN-12v2 polypeptide has been shown to comprise four glycosylationsite according to the Motif algorithm (Genetics Computer Group, Inc.).As discussed more specifically herein, protein glycosylation is thoughtto serve a variety of functions including: augmentation of proteinfolding, inhibition of protein aggregation, regulation of intracellulartrafficking to organelles, increasing resistance to proteolysis,modulation of protein antigenicity, and mediation of intercellularadhesion.

In preferred embodiments, the following asparagine glycosylation sitepolypeptide is encompassed by the present invention: RVVPLNQSFTEKYA (SEQID NO:87), IEATYNRTLIGCQT (SEQ ID NO:102), ALLDLNASGTMSIQ (SEQ IDNO:103), and/or SYLWNTTLL (SEQ ID NO:104). Polynucleotides encoding thispolypeptide are also provided. The present invention also encompassesthe use of the CAN-12v2 asparagine glycosylation site polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

The CAN-12v2 polypeptide has been shown to comprise one amidation siteaccording to the Motif algorithm (Genetics Computer Group, Inc.). Theprecursor of hormones and other active peptides which are C-terminallyamidated is always directly followed by a glycine residue which providesthe amide group, and most often by at least two consecutive basicresidues (Arg or Lys) which generally function as an active peptideprecursor cleavage site. Although all amino acids can be amidated,neutral hydrophobic residues such as Val or Phe are good substrates,while charged residues such as Asp or Arg are much less reactive. Aconsensus pattern for amidation sites is the following: x-G-[RK]-[RK],wherein “X” represents the amidation site. Additional informationrelating to asparagine glycosylation may be found in reference to thefollowing publications, which are hereby incorporated by referenceherein: Kreil G., Meth. Enzymol. 106:218-223(1984); and Bradbury A. F.,Smyth D. G., Biosci. Rep. 7:907-916(1987).

In preferred embodiments, the following amidation site polypeptide isencompassed by the present invention: VWRPEEGRRSLRPC (SEQ ID NO:88).Polynucleotides encoding these polypeptides are also provided. Thepresent invention also encompasses the use of this CAN-12v2 amidationsite polypeptide as an immunogenic and/or antigenic epitope as describedelsewhere herein.

The CAN-12v2 polypeptide has been shown to comprise one RGD cellattachment site domain according to the Motif algorithm (GeneticsComputer Group, Inc.). The sequence Arg-Gly-Asp, found in fibronectin,is crucial for its interaction with its cell surface receptor, anintegrin. What has been called the ‘RGD’ tripeptide is also found in thesequences of a number of other proteins, where it has been shown to playa role in cell adhesion. Non-limiting examples of these proteins are thefollowing: some forms of collagens, fibrinogen, vitronectin, vonWillebrand factor (VWF), snake disintegrins, and slime mold discoidins.The ‘RGD’ tripeptide is also found in other proteins where it may servethe same purpose. A consensus pattern for RGD cell attachment sites isthe following: R-G-D. Additional information relating to RGD cellattachment site domains may be found in reference to the followingpublications, which are hereby incorporated by reference herein:Ruoslahti E., Pierschbacher M. D., Cell 44:517-518(1986); and d'Souza S.E., Ginsberg M. H., Plow E. F., Trends Biochem. Sci. 16:246-250(1991).

In preferred embodiments, the following RGD cell attachment site domainpolypeptide is encompassed by the present invention: LIRQRRGDVWHAE (SEQID NO:89). Polynucleotides encoding these polypeptides are alsoprovided. The present invention also encompasses the use of this RGDcell attachment site domain polypeptide as an immunogenic and/orantigenic epitope as described elsewhere herein.

In confirmation of the CAN-12v2 polypeptide being a calpain, it has beenshown to comprise one EF-hand calcium-binding domain according to theMotif algorithm (Genetics Computer Group, Inc.). Many calcium-bindingproteins belong to the same evolutionary family and share a type ofcalcium-binding domain known as the EF-hand. This type of domainconsists of a twelve residue loop flanked on both side by a twelveresidue alpha-helical domain. In an EF-hand loop the calcium ion iscoordinated in a pentagonal bipyramidal configuration. The six residuesinvolved in the binding are in positions 1, 3, 5, 7, 9 and 12; theseresidues are denoted by X, Y, Z, −Y, −X and −Z. The invariant Glu or Aspat position 12 provides two oxygens for liganding Ca (bidentate ligand).Several representative proteins containing EF-hand regions are providedbelow: For each type of protein, the total number of EF-hand regionsknown or supposed to exist are provided in parenthesis: Aequorin andRenilla luciferin binding protein (LBP) (Ca=3); Alpha actinin (Ca=2);Calbindin (Ca=4); Calcineurin B subunit (protein phosphatase 2Bregulatory subunit) (Ca=4); Calcium-binding protein from Streptomyceserythraeus (Ca=3?); Calcium-binding protein from Schistosoma mansoni(Ca=2?); Calcium-binding proteins TCBP-23 and TCBP-25 from Tetrahymenathermophila (Ca=4?); Calcium-dependent protein kinases (CDPK) fromplants (Ca=4); Calcium vector protein from amphoxius (Ca=2); Calcyphosin(thyroid protein p24) (Ca=4?); Calmodulin (Ca=4, except in yeast whereCa=3); Calpain small and large chains (Ca=2); Calretinin (Ca=6);Calcyclin (prolactin receptor associated protein) (Ca=2); Caltractin(centrin) (Ca=2 or 4); Cell Division Control protein 31 (gene CDC31)from yeast (Ca=2?); Diacyiglycerol kinase (EC 2.7.1.107) (DGK) (Ca=2);FAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) frommammals (Ca=1); Fimbrin (plastin) (Ca-2); Flagellar calcium-bindingprotein (1f8) from Trypanosoma cruzi (Ca=1 or 2); Guanylate cyclaseactivating protein (GCAP) (Ca=3); Inositol phospholipid-specificphospholipase C isozymes gamma-1 and delta-1 (Ca=2) [10]; Intestinalcalcium-binding protein (ICaBPs) (Ca=2); MIF related proteins 8 (MRP-8or CFAG) and 14 (MRP-14) (Ca=2); Myosin regulatory light chains (Ca=1);Oncomodulin (Ca=2); Osteonectin (b)asement membrane protein BM-40)(SPARC) and proteins that contains an ‘osteonectin’ domain (QR1, matrixglycoprotein SC1) (Ca=1); Parvalbumins alpha and beta (Ca=2); Placentalcalcium-binding protein (18a2) (nerve growth factor induced protein 42a)(p9k) (Ca=2); Recoverins (visinin, hippocalcin, neurocalcin, S-modulin)(Ca=2 to 3); Reticulocalbin (Ca=4); S-100 protein, alpha and beta chains(Ca=2); Sarcoplasmic calcium-binding protein (SCPs) (Ca=2 to 3); Seaurchin proteins Spec 1 (Ca=4), Spec 2 (Ca=4?), Lps-1 (Ca=8);Serine/threonine protein phosphatase rdgc (EC 3.1.3.16) from Drosophila(Ca=2); Sorcin V19 from hamster (Ca=2); Spectrin alpha chain (Ca=2);Squidulin (optic lobe calcium-binding protein) from squid (Ca=4); andTroponins C; from skeletal muscle (Ca=4), from cardiac muscle (Ca=3),from arthropods and molluscs (Ca=2).

A consensus pattern for EF hand calcium binding domains is thefollowing: 1 2 3  4     5  6     7  8   9   10 12   13X   Y        Z       -Y     -X     -ZD-x-[DNS]-{ILVFYW}-[DENSTG]-[DNQGHRK]-{GP}-[LIVMC]-[DENQSTAGC]-x(2)-[DE]-[LIVMFYW],wherein X, Y, Z, −Y, −X, and −Z are as defined above, and wherein “x”represents any amino acid. Amino acid residues within the consensus atpositions 1 (X), 3 (Y) and 12 (−Z) are the most conserved. The 6thresidue in an EF-hand loop is in most cases a Gly.

Additional information relating to RGD cell attachment site domains maybe found in reference to the following publications, which are herebyincorporated by reference herein: Kawasaki H., Kretsinger R. H., ProteinProf. 2:305490(1995); Kretsinger R. H., Cold Spring Harbor Symp. Quant.Biol. 52:499-510(1987); Moncrief N. D., Kretsinger R. H., Goodman M., J.Mol. Evol. 30:522-562(1990); Nakayama S., Moncrief N. D., Kretsinger R.H., J. Mol. Evol. 34:416448(1992); Heizmann C. W., Hunziker W., TrendsBiochem. Sci. 16:98-103(1991); Kligman D., Hilt D. C., Trends Biochem.Sci. 13:437-443(1988); Strynadka N. C. J., James M. N. G., Annu. Rev.Biochem. 58:951-98(1989); Haiech J., Sallantin J., Biochimie67:555-560(1985); Chauvaux S., Beguin P., Aubert J.-P., Bhat K. M., GowL. A., Wood T. M., Bairoch A., Biochem. J. 265:261-265(1990); BairochA., Cox J. A., FEBS Lett. 269:454-456(1990).

In preferred embodiments, the following EF-hand calcium binding domainpolypeptide is encompassed by the present invention:ILALLDLNASGTMSIQEFRDLWK (SEQ ID NO:90). Polynucleotides encoding thesepolypeptides are also provided. The present invention also encompassesthe use of this EF-hand calcium binding domain polypeptide as animmunogenic and/or antigenic epitope as described elsewhere herein.

In further confirmation of the CAN-12v2 polypeptide being a calpain, ithas been shown to comprise one eukaryotic thiol (cysteine) proteaseactive site domain according to the Motif algorithm (Genetics ComputerGroup, Inc.). Eukaryotic thiol proteases (EC 3.4.22.-) are a family ofproteolytic enzymes which contain an active site cysteine. Catalysisproceeds through a thioester intermediate and is facilitated by a nearbyhistidine side chain; an asparagine completes the essential catalytictriad. Non-limiting examples of proteases which are known to belong tothis family are provided below: Vertebrate lysosomal cathepsins B (EC3.4.22.1), H (EC 3.4.22.16), L (EC 3.4.22.15), and S (EC 3.4.22.27);Vertebrate lysosomal dipeptidyl peptidase I (EC 3.4.14.1) (also known ascathepsin C); Vertebrate calpains (EC 3.4.22.17) (Calpains areintracellular calcium-activated thiol protease that contain both aN-terminal catalytic domain and a C-terminal calcium-binding domain;Mammalian cathepsin K, which seems involved in osteoclastic boneresorption; Human cathepsin O; Bleomycin hydrolase (An enzyme thatcatalyzes the inactivation of the antitumor drug BLM (a glycopeptide);Plant enzymes: barley aleurain (EC 3.4.22.16), EP-B1/B4; kidney beanEP-C1, rice bean SH-EP; kiwi fruit actinidin (EC 3.4.22.14); papayalatex papain (EC 3.4.22.2), chymopapain (EC 3.4.22.6), caricain (EC3.4.22.30), and proteinase IV (EC 3.4.22.25); pea turgor-responsiveprotein 15A; pineapple stem bromelain (EC 3.4.22.32); rape COT44; riceoryzain alpha, beta, and gamma; tomato low-temperature induced,Arabidopsis thaliana A494, RD19A and RD21 A; House-dust mites allergensDerP 1 and EurM1; Cathepsin B-like proteinases from the wormsCaenorhabditis elegans (genes gcp-1, cpr-3, cpr-4, cpr-5 and cpr-6),Schistosoma mansoni (antigen SM31) and Japonica (antigen SJ31),Haemonchus contortus (genes AC-1 and AC-2), and Ostertagia ostertagi(CP-1 and CP-3); Slime mold cysteine proteinases CP1 and CP2; Cruzipainfrom Trypanosoma cruzi and brucei; Throphozoite cysteine proteinase(TCP) from various Plasmodium species; Proteases from Leishmaniamexicana, Theileria annulata and Theileria parva; Baculovirusescathepsin-like enzyme (v-cath); Drosophila small optic lobes protein(gene sol), a neuronal protein that contains a calpain-like domain;Yeast thiol protease BLH1/JYCP1/LAP3; and Caenorhabditis eleganshypothetical protein C06G4.2, a caipain-like protein; Two bacterialpeptidases are also part of this family—Aminopeptidase C fromLactococcus lactis (gene pepC), and Thiol protease tpr fromPorphyromonas gingivalis.

A consensus pattern for eukaryotic thiol (cysteine) protease active sitedomains is the following: Q-x(3)-[GE]-x-C-[YW]-x(2)-[STAGC]-[STAGCV],wherein C is the active site residue, and “x” represents any amino acid.The residue in position 4 of the pattern is almost always cysteine; theonly exceptions are calpains (Leu), bleomycin hydrolase (Ser) and yeastYCP1 (Ser); while the residue in position 5 of the pattern is always Glyexcept in papaya protease IV where it is Glu.

An additional consensus pattern for eukaryotic thiol (cysteine) proteaseactive site domains is the following:[LIVMGSTAN]-x-H-[GSACE]-[LIVM]-x-[LIVMAT](2)-G-x-[GSADNH], wherein H isthe active site residue, and “x” represents any amino acid.

An additional consensus pattern for eukaryotic thiol (cysteine) proteaseactive site domains is the following:[FYCH]-[WI]-[LIVT]-x-[KRQAG]-N-[ST]-W-x(3)-[FYW]-G-x(2)-G-[LFYW]-[LIVMFYG]-x-[LIVMF],wherein N is the active site residue, and “x” represents any amino acid.

Additional information relating to for eukaryotic thiol (cysteine)protease active site domains may be found in reference to the followingpublications, which are hereby incorporated by reference herein: DufourE., Biochimie 70:1335-1342(1988); Kirschke H., Barrett A. J., RawlingsN. D., Protein Prof. 2:1587-1643(1995); Shi G.-P., Chapman H. A., BhairiS. M., Deleeuw C., Reddy V. Y., Weiss S. J., FEBS Lett.357:129-134(1995); Velasco G., Ferrando A. A., Puente X. S., Sanchez L.M., Lopez-Otin C., J. Biol. Chem. 269:27136-27142(1994); Chapot-ChartierM. P., Nardi M., Chopin M. C., Chopin A., Gripon J. C., Appl. Environ.Microbiol. 59:330-333(1993); Higgins D. G., McConnell D. J., Sharp P.M., Nature 340:604-604(1989); Rawlings N. D., Barrett A. J., Meth.Enzymol. 244:461-486(1994), andhttp://www.expasy.ch/cgi-bin/lists?peptidas.txt, which are herebyincorporated by reference in their entirety herein.

In preferred embodiments, the following for eukaryotic thiol (cysteine)protease active site domain polypeptide is encompassed by the presentinvention: RLDLCQGIVGDCWFLAALQALA (SEQ ID NO:91). Polynucleotidesencoding these polypeptides are also provided. The present inventionalso encompasses the use of this for eukaryotic thiol (cysteine)protease active site domain polypeptide as an immunogenic and/orantigenic epitope as described elsewhere herein.

As referenced elsewhere herein, calpains are organized in domains. As apoint of reference, the larger catalytic subunit of the bestcharacterized m-calpain is organized in four domains (I-IV)(Hosfield etal., Crystal structure of calpain reveals the structural basis forCa(2+)-dependent protease activity and a novel mode of enzymeactivation. EMBO J 18:6880-9, 1999; Strobl et al., The crystal structureof calcium-free human m-calpain suggests an electrostatic switchmechanism for activation by calcium. Proc Natl Acad Sci U S A.97:588-92, 2000). The N-terminal domain I contains an alpha helicalregion. Domain II contains the catalytic active domain with the activesite amino acids. Domain m contains the linker between the Ca2+ bindingdomain in domain IV to the active site domain II.

The CAN-12v2 calpain of the present invention has the same domain I andII as the CAN-12 calpain, but differs in domains III and IV. TheN-terminal domain I consists of residues Met1-Arg20. Domain II of theCAN-12v2 calpain (Ala2l-Lys333) contain the catalytic active siteresidue acids (Cys101, His254 and Asn 278). As can be seen in thesequence alignments (FIGS. 2A-E), there is high amino acid sequencehomology in the amino acid residues bracketing the active site aminoacids. Combined domains I and II of the calpains of the presentinvention are 42-45% homologous to m-calpain.

The CAN-12v2 calpain of the present invention, have the same domain Iand II, although they differ in composition and content of domains IIIand IV. The CAN-12 and CAN-12v2 calpains contain both the linker (domainIII) and C-terminal domain IV. The “linker” domain also containsresidues Met426, Asn427 and Lys428 of SEQ ID NO56).

The present invention also provides a three-dimensional homology modelof the CAN-12v2 polypeptide (see FIG. 11). The three-dimensionalhomology model of the CAN-12 polypeptide may also be applicable to theCAN-12v2 polypeptide. Although the CAN-12 polypeptide sequence isdifferent than the CAN-12v2. polypeptide sequence, the fact that domainI and II are substantially the same suggests the homology model ofCAN-12 may be used for designing potential ligands (including agonistsand/or antagonists) for the CAN-12v2 polypeptide. A three-dimensionalhomology model can be constructed on the basis of the known structure ofa homologous protein (Greer et al, 1991, Lesk, et al, 1992, Cardozo, etal, 1995, Yuan, et al, 1995). The homology model of the CAN-12v2polypeptide, corresponding to amino acid residues 12 to 428 and fromamino acid residues 543 to 639 of SEQ ID NO:56, was based upon thehomologous structure of CAN2, a m-calpain family member (Strobl et al,2000; hCAN2; Genbank Accession No. gi|7546423; SEQ ID NO:11) and isdefined by the set of structural coordinates set forth in Table Vherein. Note that amino acids 429 to 542 of SEQ ID NO:56 were omittedfrom the homology model. As a result, the amino acid residue numbers inTable V do not correspond to the amino acid residue numbers as providedin SEQ ID NO:56 (FIGS. 9A-C). Rather, the amino acid residue numbers inTable V for amino acid residues 12 to 428 correspond to amino acidresidues 12 to 428 of SEQ ID NO:56 (FIGS. 9A-C), while amino acidresidue numbers in Table V for amino acid residues 429 to 512 correspondto amino acid residues 543 to 639 of SEQ ID NO:56 (FIGS. 9A-C).

A description of the headings in Table V are as follows: “Atom No”refers to the atom number within the CAN-12v2 homology model; “Atomname” refers to the element whose coordinates are measured, the firstletter in the column defines the element; “Residue” refers to the aminoacid within which the atom resides, and the provided number after theamino acid refers to the amino acid number of the “residue”; “X Coord”,“Y Coord”, and “Z Coord” structurally define the atomic position of theelement measured in three dimensions.

The CAN-12v2 homology model of the present invention may provide onebasis for designing rational stimulators (agonists) and/or inhibitors(antagonists) of one or more of the biological functions of CAN-12v2, orof CAN-12v2 mutants having altered specificity (e.g., molecularlyevolved CAN-12v2 polypeptides, engineered site-specific CAN-12v2mutants, CAN-12v2 allelic variants, etc.).

Homology models are not only useful for designing rational agonistsand/or antagonists, but are also useful in predicting the function of aparticular polypeptide. The functional predictions from homology modelsare typically more accurate than the functional attributes derived fromtraditional polypeptide sequence homology alignments (e.g., CLUSTALW),particularly when the three dimensional structure of a relatedpolypeptide is known (e.g., m-calpain family member CAN2 protein;Genbank Accession No. gi|7546423; SEQ ID NO:11). The increasedprediction accuracy is based upon the fact that homology modelsapproximate the three-dimensional structure of a protein, while homologybased alignments only take into account the one dimension polypeptidesequence. Since the function of a particular polypeptide is determinednot only by its primary, secondary, and tertiary structure, functionalassignments derived solely upon homology alignments using the onedimensional protein sequence may be less reliable. A 3-dimensional modelcan be constructed on the basis of the known structure of a homologousprotein (Greer et al, 1991, Lesk, et al, 1992, Cardozo, et al, 1995,Yuan, et al, 1995).

Prior to developing a homology model, those of skill in the art wouldappreciate that a template of a known protein, or model protein, mustfirst be identified which will be used as a basis for constructing thehomology model for the protein of unknown structure (query template). Inthe case of the CAN-12v2 polypeptide of the present invention, the modelprotein template used in constructing the CAN-12v2 homology model wasthe m-calpain family member CAN2 protein; Genbank Accession No.gi|7546423; SEQ ID NO:11).

Identifying a template can be accomplished using pairwise alignment ofprotein sequences using such programs as FASTA (Pearson, et al 1990) andBLAST (Altschul, et al, 1990). In cases where sequence similarity ishigh (greater than 30%), such pairwise comparison methods may beadequate for identifying an appropriate template. Likewise, multiplesequence alignments or profile-based methods can be used to align aquery sequence to an alignment of multiple (structurally andbiochemically) related proteins. When the sequence similarity is low,more advanced techniques may be used. Such techniques, include, forexample, protein fold recognition (protein threading; Hendlich, et al,1990), where the compatibility of a particular polypeptide sequence withthe 3-dimensional fold of a potential template protein is gauged on thebasis of a knowledge-based potential.

Following the initial sequence alignment, the second step would be tooptimally align the query template to the model template by manualmanipulation and/or by the incorporation of features specific to thepolypeptides (e.g., motifs, secondary structure predictions, and allowedconservations). Preferably, the incorporated features are found withinboth the model and query template.

The third step would be to identify structurally conserved regions thatcould be used to construct secondary core structure (Sali, et al, 1995).Loops could be added using knowledge-based techniques, and by performingforcefield calculations (Sali, et al, 1995).

In order to recognize errors in a three-dimensional structure, knowledgebased mean fields can be used to judge the quality of protein folds(Sippl 1993). The methods can be used to recognize misfolded structuresas well as faulty parts of structural models. The technique generates anenergy graph where the energy distribution for a given protein fold isdisplayed on the y-axis and residue position in the protein fold isdisplayed on the x-axis. The knowledge based mean fields compose a forcefield derived from a set of globular protein structures taken as asubset from the Protein Data Bank (Bernstein et. al. 1977). To analyzethe quality of a model the energy distribution is plotted and comparedto the energy distribution of the template from which the model wasgenerated. FIG. 13 shows the energy graph for the CAN-12.v2 model(dotted line) and the template (1dkv, m-calpain) from which the modelwas generated. This graph supports the motif and sequence alignments inconfirming that the three dimensional structure coordinates of CAN-12.v2are an accurate and useful representation for the polypeptide.

The term “structure coordinates” refers to Cartesian coordinatesgenerated from the building of a homology model. In this invention, thehomology model of residues 12 to 525 of CAN-12v2 was derived fromgenerating a sequence alignment with m-calpain (Strobl et al, 2000;hCAN2; Genbank Accession No. gi|7546423; SEQ ID NO:11) using theCOMPOSER suite of software within SYBYL6.6 (Tripos Associates, St.Louis, Mo.) and then generating the backbone and side chainconformations. In the original crystal structure (pdb code 1dkv) as wellas the crystal structure reported elsewhere (Hosfield et al, 1999), theactive site of the enzyme comprising a cysteine, a histidine and anasparagine residue was not “formed”. The helix that contains the activesite C101 was altered by moving the helix down one pitch so that theactive site geometry could match that found in Papain (pdb code 1b4).This modified structure of human m-calpain was used as the template forconstruction of the homology model (illustrated in FIG. 11 herein).

The skilled artisan would appreciate that a set of structure coordinatesfor a protein represents a relative set of points that define a shape inthree dimensions. Thus, it is possible that an entirely different set ofcoordinates could define a similar or identical shape. Moreover, slightvariations in the individual coordinates, as emanate from the generationof similar homology models using different alignment templates (i.e.,other than the m-calpain (Strobl et al, 2000; hCAN2; Genbank AccessionNo. gi|7546423; SEQ ID NO:11), and/or using different methods ingenerating the homology model, will likely have minor effects on theoverall shape. Variations in coordinates may also be generated becauseof mathematical manipulations of the structure coordinates. For example,the structure coordinates set forth in Table V could be manipulated byfractionalization of the structure coordinates; integer additions, orinteger subtractions to sets of the structure coordinates, inversion ofthe structure coordinates or any combination of the above.

Therefore, various computational analyses are necessary to determinewhether a template molecule or a portion thereof is sufficiently similarto all or part of a query template (e.g., CAN-12v2) in order to beconsidered the same. Such analyses may be carried out in currentsoftware applications, such as SYBYL version 6.6 or INSIGHTII (MolecularSimulations Inc., San Diego, Calif.) version 2000 and as described inthe accompanying User's Guides.

Using the superimposition tool in the program SYBYL, comparisons can bemade between different structures and different conformations of thesame structure. The procedure used in SYBYL to compare structures isdivided into four steps: 1) load the structures to be compared; 2)define the atom equivalencies in these structures; 3) perform a fittingoperation; and 4) analyze the results. Each structure is identified by aname. One structure is identified as the target (i.e., the fixedstructure); the second structure (i.e., moving structure) is identifiedas the source structure. The atom equivalency within SYBYL is defined byuser input. For the purpose of this invention, we will define equivalentatoms as protein backbone atoms (N, Cα, C and O) for all conservedresidues between the two structures being compared. We will alsoconsider only rigid fitting operations. When a rigid fitting method isused, the working structure is translated and rotated to obtain anoptimum fit with the target structure. The fitting operation uses analgorithm that computes the optimum translation and rotation to beapplied to the moving structure, such that the root mean squaredifference of the fit over the specified pairs of equivalent atoms is anabsolute minimum. This number, given in angstroms, is reported by theSYBYL program. For the purpose of the present invention, any homologymodel of a CAN-12v2 that has a root mean square deviation of conservedresidue backbone atoms (N, Cα, C, O) of less than 3.0 A whensuperimposed on the relevant backbone atoms described by structurecoordinates listed in Table V are considered identical. More preferably,the root mean square deviation for the CAN-12v2 polypeptide is less than2.0 Å.

The homology model of the present invention is useful for thestructure-based design of modulators of the CAN-12v2 biologicalfunction, as well as mutants with altered biological function and/orspecificity.

In accordance with the structural coordinates provided in Table V andthe three dimensional homology model of CAN-12v2, the CAN-12v2polypeptide has been shown to comprise a an active site region embodiedby the following amino acids: from about amino acid R90 to about aminoacid C94, from about amino acid G99 to about amino acid L104, from aboutamino acid Q196 to about amino acid S198, from about amino acid M210 toabout amino acid I212, from about amino acid G236 to about amino acidH240, from about amino acid E252 to about amino acid Y256, from aboutamino acid N278 to about amino acid K282, from about amino acid V328 toabout amino acid V331 of SEQ ID NO:56 (FIGS. 8A-C). In this context, theterm “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acids more in either the N— or C-terminal direction of the abovereferenced amino acids.

Also more preferred are polypeptides comprising all or any part of theCAN-12v2 active site domain, or a mutant or homologue of saidpolypeptide or molecular complex. By mutant or homologue of the moleculeis meant a molecule that has a root mean square deviation from thebackbone atoms of said CAN-12v2 amino acids of not more than about 4.5Angstroms, and preferably not more than about 3.5 Angstroms.

In preferred embodiments, the following CAN-12v2 active site domainpolypeptide is encompassed by the present invention:RLDLCQGIVGDCWFLAALQALALHQDILSRVVPLNQSFTEKYAGIFRFWFWHYGNWVPVVIDDRLPVNEAGQLVFVSSTYKNLFWGALLEKAYAKLSGSYEDLQSGQVSEALVDFTGGVTMTINLAEAHGNLWDILEATYNRTLIGCQTHSGEKILENGLVEGHAYTLTGIRKVTCKHRPEYLVKLRNPWGKVEWKGDWSDSSSKWELLSPKEKILLLRKDNDGEFWMTLQDFKTHFVLLV (SEQ ID NO:93). Polynucleotides encoding thispolypeptide are also provided. The present invention also encompassesthe use of the CAN-12v2 active site domain polypeptide as an immunogenicand/or antigenic epitope as described elsewhere herein.

The present invention also encompasses polypeptides comprising at leasta portion of the CAN-12v2 active site domain (SEQ ID NO: 93). Suchpolypeptides may correspond, for example, to the N— and/or C-terminaldeletions of the active site domain.

In preferred embodiments, the following N-terminal CAN-12v2 active sitedomain deletion polypeptides are encompassed by the present invention:R1-V242, L2-V242, D3-V242, L4-V242, C5-V242, Q6-V242, G7-V242, I8-V242,V9-V242, G10-V242, C12-V242, W13-V242, F14-V242, L15-V242, A16-V242,A17-V242, L18-V242, Q19-V242, A20-V242, L21-V242, A22-V242, L23-V242,H24-V242, Q25-V242, D26-V242, I27-V242, L28-V242, S29-V242, R30-V242,V31-V242, V32-V242, P33-V242, L34-V242, N35-V242, Q36-V242, S37-V242,F38-V242, T39-V242, E40-V242, K41-V242, Y42-V242, A43-V242, G44-V242,I45-V242, F46-V242, R47-V242, F48-V242, W49-V242, F50-V242, W51-V242,H52-V242, Y53-V242, G54-V242, N55-V242, W56-V242, V57-V242, P58-V242,V59-V242, V60-V242, I61-V242, D62-V242, D63-V242, R64-V242, L65-V242,P66-V242, V67-V242, N68-V242, E69-V242, A70-V242, G71-V242, Q72-V242,L73-V242, V74-V242, F75-V242, V76-V242, S77-V242, S78-V242, T79-V242,Y80-V242, K81-V242, N82-V242, L83-V242, F84-V242, W85-V242, G86-V242,A87-V242, L88-V242, L89-V242, E90-V242, K91-V242, A92-V242, Y93-V242,A94-V242, K95-V242, L96-V242, S97-V242, G98-V242, S99-V242, Y100-V242,E101-V242, D102-V242, L103-V242, Q104-V242, S105-V242, G106-V242,Q107-V242, V108-V242, S109-V242, E110-V242, A111-V242, L112-V242,V113-V242, D114-V242, F115-V242, T116-V242, G117-V242, G118-V242,V119-V242, T120-V242, M121-V242, T122-V242, I123-V242, N124-V242,L125-V242, A126-V242, E127-V242, A128-V242, H129-V242, G130-V242,N131-V242, L132-V242, W133-V242, D134-V242, I135-V242, L136-V242,I137-V242, E138-V242, A139-V242, T140-V242, Y141-V242, N142-V242,R143-V242, T144-V242, L145-V242, I146-V242, G147-V242, C148-V242,Q149-V242, T150-V242, H151-V242, S152-V242, G153-V242, E154-V242,K155-V242, I156-V242, L157-V242, E158-V242, N159-V242, G160-V242,L161-V242, V162-V242, E163-V242, G164-V242, H165-V242, A166-V242,Y167-V242, T168-V242, L169-V242, T170-V242, G171-V242, I172-V242,R173-V242, K174-V242, V175-V242, T176-V242, C177-V242, K178-V242,H179-V242, R180-V242, P181-V242, E182-V242, Y183-V242, L184-V242,V185-V242, K186-V242, L187-V242, R188-V242, N189-V242, P190-V242,W191-V242, G192-V242, K193-V242, V194-V242, E195-V242, W196-V242,K197-V242, G198-V242, D199-V242, W200-V242, S201-V242, D202-V242,S203-V242, S204-V242, S205-V242, K206-V242, W207-V242, E208-V242,L209-V242, L210-V242, S211-V242, P212-V242, K213-V242, E214-V242,K215-V242, I216-V242, L217-V242, L218-V242, L219-V242, R220-V242,K221-V242, D222-V242, N223-V242, D224-V242, G225-V242, E226-V242,F227-V242, W228-V242, M229-V242, T230-V242, L231-V242, Q232-V242,D233-V242, F234-V242, K235-V242, and/or T236-V242 of SEQ ID NO:93.Polynucleotide sequences encoding these polypeptides are also provided.The present invention also encompasses the use of these N-terminalCAN-12v2 active site domain deletion polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal CAN-12v2 active sitedomain deletion polypeptides are encompassed by the present invention:R1-V242, R1-L241, R1-L240, R1-V239, R1-F238, R1-H237, R1-T236, R1-K235,R1-F234, R1-D233, R1-Q232, R1-L231, R1-T230, R1-M229, R1-W228, R1-F227,R1-E226, R1-G225, R1-D224, R1-N223, R1-D222, R1-K221, R1-R220, R1-L219,R1-L218, R1-L217, R1-I216, R1-K215, R1-E214, R1-K213, R1-P212, R1-S211,R1-L210, R1-L209, R1-E208, R1-W207, R1-K206, R1-S205, R1-S204, R1-S203,R1-D202, R1-S201, R1-W200, R1-D199, R1-G198, R1-K197, R1-W196, R1-E195,R1-V194, R1-K193, R1-G192, R1-W191, R1-P190, R1-N189, R1-R188, R1-L187,R1-K186, R1-V185, R1-L184, R1-Y183, R1-E182, R1-P181, R1-R180, R1-H179,R1-K178, R1-C177, R1-T176, R1-V175, R1-K174, R1-R173, R1-I172, R1-G171,R1-T170, R1-L169, R1-T168, R1-Y167, R1-A166, R1-H165, R1-G164, R1-E163,R1-V162, R1-L161, R1-G160, R1-N159, R1-E158, R1-L157, R1-I156, R1-K155,R1-E154, R1-G153, R1-S152, R1-H151, R1-T150, R1-Q149, R1-C148, R1-G147,R1-I146, R1-L145, R1-T144, R1-R143, R1-N142, R1-Y141, R1-T140, R1-A139,R1-E138, R1-I137, R1-L136, R1-I135, R1-D134, R1-W133, R1-L132, R1-N131,R1-G130, R1-H129, R1-A128, R1-E127, R1-A126, R1-L125, R1-N124, R1-I123,R1-T122, R1-M121, R1-T120, R1-V119, R1-G118, R1-G117, R1-T116, R1-F115,R1-D114, R1-V113, R1-L112, R1-A111, R1-E110, R1-S109, R1-V108, R1-Q107,R1-G106, R1-S105, R1-Q104, R1-L103, R1-D102, R1-E101, R1-Y100, R1-S99,R1-G98, R1-S97, R1-L96, R1-K95, R1-A94, R1-Y93, R1-A92, R1-K91, R1-E90,R1-L89, R1-L88, R1-A87, R1-G86, R1-W85, R1-F84, R1-L83, R1-N82, R1-K81,R1-Y80, R1-T79, R1-S78, R1-S77, R1-V76, R1-F75, R1-V74, R1-L73, R1-Q72,R1-G71, R1-A70, R1-E69, R1-N68, R1-V67, R1-P66, R1-L65, R1-R64, R1-D63,R1-D62, R1-I61, R1-V60, R1-V59, R1-P58, R1-V57, R1-W56, R1-N55, R1-G54,R1-Y53, R1-H52, R1-W51, R1-F50, R1-W49, R1-F48, R1-R47, R1-F46, R1-I45,R1-G44, R1-A43, R1-Y42, R1-K41, R1-E40, R1-T39, R1-F38, R1-S37, R1-Q36,R1-N35, R1-L34, R1-P33, R1-V32, R1-V31, R1-R30, R1-S29, R1-L28, R1-I27,R1-D26, R1-Q25, R1-H24, R1-L23, R1-A22, R1-L21, R1-A20, R1-Q19, R1-L18,R1-A17, R1-A16, R1-L15, R1-F14, R1-W13, R1-C12, R1-D11, R1-G10, R1-V9,R1-I8, and/or R1-G7 of SEQ ID NO:93. Polynucleotide sequences encodingthese polypeptides are also provided. The present invention alsoencompasses the use of these C-terminal CAN-12v2 active site domaindeletion polypeptides as immunogenic and/or antigenic epitopes asdescribed elsewhere herein.

Alternatively, such polypeptides may comprise polypeptide sequencescorresponding, for example, to internal regions of the CAN-12v2 activesite domain (e.g., any combination of both N— and C-terminal CAN-12v2active site domain deletions) of SEQ ID NO:93. For example, internalregions could be defined by the equation NX to CX, where NX refers toany N-terminal amino acid position of the CAN-12v2 active site domain(SEQ ID NO:93), and where CX refers to any C-terminal amino acidposition of the CAN-12v2 active site domain (SEQ ID NO:93).Polynucleotides encoding these polypeptides are also provided. Thepresent invention also encompasses the use of these polypeptides as animmunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following CAN-12v2 active site domainamino acid substitutions are encompassed by the present invention:wherein R90 is substituted with either an A, C, D, E, F, G, H, I, K, L,M, N, P, Q, S, T, V, W, or Y; wherein L91 is substituted with either anA, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein D92is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W, or Y; wherein L93 is substituted with either an A, C, D, E,F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein C94 issubstituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein Q95 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein G96 is substitutedwith either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein I97 is substituted with either an A, C, D, E, F, G, H, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein V98 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; whereinG99 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y; wherein D100 is substituted with either an A, C, E,F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein C101 issubstituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein W102 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein F103 is substitutedwith either an A, C, D, E, G. H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein L104 is substituted with either an A, C, D, E, F, G, H, I, K,M, N, P, Q, R, S, T, V, W, or Y; wherein A105 is substituted with eithera C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinA106 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y; wherein L107 is substituted with either an A, C, D,E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein Q108 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S,T, V, W, or Y; wherein A109 is substituted with either a C, D, E, F, G,H, I, K, L, M, N, P, Q, P, S, T, V, W, or Y; wherein L110 is substitutedwith either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY; wherein A111 is substituted with either a C, D, E, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein L112 is substituted with eitheran A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; whereinH113 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein Q114 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein D115 issubstituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein I116 is substituted with either an A, C, D, E, F,G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L117 is substitutedwith either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY; wherein S118 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, T, V, W, or Y; wherein R119 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; whereinV120 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, W, or Y; wherein V121 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein P122 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S,T, V, W, or Y; wherein L123 is substituted with either an A, C, D, E, F,G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein N124 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; wherein Q125 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, R, S, T, V, W, or Y; wherein S126 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; whereinF127 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein T128 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein E129 issubstituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein K130 is substituted with either an A, C, D, E, F,G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y131 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, orW; wherein A132 is substituted with either a C, D, E, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein G133 is substituted with eitheran A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinI134 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein F135 is substituted with either an A, C,D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R136 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S,T, V, W, or Y; wherein F137 is substituted with either an A, C, D, E, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W138 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, orY; wherein F139 is substituted with either an A, C, D, E, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein W140 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; whereinH141 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein Y142 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein G143 issubstituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein N144 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein W145 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, orY; wherein V146 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, T, W, or Y; wherein P147 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; whereinV148 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, W, or Y; wherein V149 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein I150 issubstituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein D151 is substituted with either an A, C, E, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D152 is substitutedwith either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein R153 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, S, T, V, W, or Y; wherein L154 is substituted with eitheran A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; whereinP155 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,Q, R, S, T, V, W, or Y; wherein V156 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein N157 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S,T, V, W, or Y; wherein E158 is substituted with either an A, C, D, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A159 is substitutedwith either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein G160 is substituted with either an A, C, D, E, F, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein Q161 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; whereinL162 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P,Q, R, S, T, V, W, or Y; wherein V163 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein F164 issubstituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein V165 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein S166 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, orY; wherein S167 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, T, V, W, or Y; wherein T168 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; whereinY169 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, V, or W; wherein K170 is substituted with either an A, C,D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N171 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S,T, V, W, or Y; wherein L172 is substituted with either an A, C, D, E, F,G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein F173 is substitutedwith either an A, C, D, F, G, H, I, K, L, M, N, P, Q, P, S, T, V, W, orY; wherein W174 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, T, V, or Y; wherein G175 is substituted with eitheran A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinA176 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y; wherein L177 is substituted with either an A, C, D,E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein L178 issubstituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S,T, V, W, or Y; wherein E179 is substituted with either an A, C, D, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K180 is substitutedwith either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, orY; wherein A181 is substituted with either a C, D, E, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein Y182 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; whereinA183 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y; wherein K184 is substituted with either an A, C, D,E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L185 issubstituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S,T, V, W, or Y; wherein S186 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein G187 is substitutedwith either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein S188 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, T, V, W, or Y; wherein Y189 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; whereinE190 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein D191 is substituted with either an A, C,E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L192 issubstituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S,T, V, W, or Y; wherein Q193 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein S194 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, orY; wherein G195 is substituted with either an A, C, D, E, F, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein Q196 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; whereinV197 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, W, or Y; wherein S198 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein E199 issubstituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein A200 is substituted with either a C, D, E, F, G,H, I, K, L, M, N, P, Q, P, S, T, V, W, or Y; wherein L201 is substitutedwith either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY; wherein V202 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, T, W, or Y; wherein D203 is substituted with eitheran A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinF204 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein T205 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein G206 issubstituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein G207 is substituted with either an A, C, D, E, F,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V208 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, orY; wherein T209 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, V, W, or Y; wherein W110 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; whereinT211 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, V, W, or Y; wherein I212 is substituted with either an A, C,D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N213 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S,T, V, W, or Y; wherein L214 is substituted with either an A, C, D, E, F,G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein A215 is substitutedwith either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein E216 is substituted with either an A, C, D, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein A217 is substituted with eithera C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinH218 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein G219 is substituted with either an A, C,D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N220 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S,T, V, W, or Y; wherein L221 is substituted with either an A, C, D, E, F,G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein W222 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, orY; wherein D223 is substituted with either an A, C, E, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein I224 is substituted with eitheran A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinL225 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P,Q, R, S, T, V, W, or Y; wherein I226 is substituted with either an A, C,D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E227 issubstituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein A228 is substituted with either a C, D, E, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T229 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, orY; wherein Y230 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, T, V, or W; wherein N231 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; whereinR232 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, S, T, V, W, or Y; wherein T233 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein L234 issubstituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S,T, V, W, or Y; wherein I235 is substituted with either an A, C, D, E, F,G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G236 is substitutedwith either an A, C, D, E, F, H, I, K, L, M, N, P, Q, P, S, T, V, W, orY; wherein C237 is substituted with either an A, D, E, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein Q238 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; whereinT239 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, V, W, or Y; wherein H240 is substituted with either an A, C,D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S241 issubstituted with either an A, C, D, E, F, G, HI I, K, L, M, N, P, Q, R,T, V, W, or Y; wherein G242 is substituted with either an A, C, D, E, F,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E243 is substitutedwith either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein K244 is substituted with either an A, C, D, E, F, G, H, I, L,M, N, P, Q, R, S, T, V, W, or Y; wherein I245 is substituted with eitheran A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinL246 is substituted with either an A, C, D, E F, G, H, I, K, M, N, P, Q,R, S, T, V, W, or Y; wherein E247 is substituted with either an A, C, D,F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N248 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S,T, V, W, or Y; wherein G249 is substituted with either an A, C, D, E, F,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L250 is substitutedwith either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY; wherein V251 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, T, W, or Y; wherein E252 is substituted with eitheran A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinG253 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein H254 is substituted with either an A, C,D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A255 issubstituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein Y256 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein T257 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, orY; wherein L258 is substituted with either an A, C, D, E, F, G, H, I, K,M, N, P, Q, R, S, T, V, W, or Y; wherein T259 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; whereinG260 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein I261 is substituted with either an A, C,D, E F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R262 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S,T, V, W, or Y; wherein K263 is substituted with either an A, C, D, F, F,G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V264 is substitutedwith either an A, C, D, E F, G, H, I, K, L, M, N, P, Q, R, S, T, W, orY; wherein T265 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, V, W, or Y; wherein C266 is substituted with eitheran A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinK267 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P,Q, R, S, T, V, W, or Y; wherein H268 is substituted with either an A, C,D, E, F, G, I, K, L, M, N, P, Q, P, S, T, V, W, or Y,; wherein R269 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S,T, V, W, or Y; wherein P270 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein E271 is substitutedwith either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein Y272 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, T, V, or W; wherein L273 is substituted with eitheran A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; whereinV274 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, W, or Y; wherein K275 is substituted with either an A, C,D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L276 issubstituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S,T, V, W, or Y; wherein R277 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein N278 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; wherein P279 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, Q, R, S, T, V, W, or Y; wherein W280 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; whereinG281 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein K282 is substituted with either an A, C,D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V283 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, W, or Y; wherein E284 is substituted with either an A, C, D, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W285 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, orY; wherein K286 is substituted with either an A, C, D, E, F, G, H, I, L,M, N, P, Q, R, S, T, V, W, or Y; wherein G287 is substituted with eitheran A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinD288 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein W289 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein S290 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,T, V, W, or Y; wherein D291 is substituted with either an A, C, E, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S292 is substitutedwith either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, orY; wherein S293 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, T, V, W, or Y; wherein S294 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; whereinK295 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P,Q, R, S, T, V, W, or Y; wherein W296 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein E297 issubstituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein L298 is substituted with either an A, C, D, E, F,G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein L299 is substitutedwith either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY; wherein S300 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, T, V, W, or Y; wherein P301 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; whereinK302 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P,Q, R, S, T, V, W, or Y; wherein E303 is substituted with either an A, C,D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K304 issubstituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, P, S,T, V, W, or Y; wherein I305 is substituted with either an A, C, D, E, F,G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L306 is substitutedwith either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY; wherein L307 is substituted with either an A, C, D, E, F, G, H, I, K,M, N, P, Q, R, S, T, V, W, or Y; wherein L308 is substituted with eitheran A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; whereinR309 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N,P, Q, S, T, V, W, or Y; wherein K310 is substituted with either an A, C,D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D311 issubstituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; wherein N312 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein D313 is substitutedwith either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein G314 is substituted with either an A, C, D, E, F, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; wherein E315 is substituted with eitheran A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinF316 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein W317 is substituted with either an A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein M318 issubstituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S,T, V, W, or Y; wherein T319 is substituted with either an A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein L320 is substitutedwith either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, orY; wherein Q321 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, R, S, T, V, W, or Y; wherein D322 is substituted with eitheran A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; whereinF323 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; wherein K324 is substituted with either an A, C,D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T325 issubstituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, V, W, or Y, wherein H326 is substituted with either an A, C, D, E, F,G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F327 is substitutedwith either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY; wherein V328 is substituted with either an A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, T, W, or Y; wherein L329 is substituted with eitheran A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; whereinL330 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P,Q, R, S, T, V, W, or Y; and/or wherein V331 is substituted with eitheran A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y of SEQ IDNO:56, in addition to any combination thereof. The present inventionalso encompasses the use of these CAN-12v2 active site domain amino acidsubstituted polypeptides as imrnunogenic and/or antigenic epitopes asdescribed elsewhere herein.

In preferred embodiments, the following CAN-12v2 active site domainconservative amino acid substitutions are encompassed by the presentinvention: wherein R90 is substituted with either a K, or H; wherein L91is substituted with either an A, I, or V; wherein D92 is substitutedwith an E; wherein L93 is substituted with either an A, I, or V; whereinC94 is a C; wherein Q95 is substituted with a N; wherein G96 issubstituted with either an A, M, S, or T; wherein I97 is substitutedwith either an A, V, or L; wherein V98 is substituted with either an A,I, or L; wherein G99 is substituted with either an A, M, S, or T;wherein D100 is substituted with an E; wherein C101 is a C; wherein W102is either an F, or Y; wherein F103 is substituted with either a W, or Y;wherein L104 is substituted with either an A, I, or V; wherein A105 issubstituted with either a G, I, L, M, S, T, or V; wherein A106 issubstituted with either a G, I, L, M, S, T, or V; wherein L107 issubstituted with either an A, I, or V; wherein Q108 is substituted witha N; wherein A109 is substituted with either a G, I, L, M, S, T, or V;wherein L110 is substituted with either an A, I, or V; wherein A111 issubstituted with either a G, I, L, M, S, T, or V; wherein L112 issubstituted with either an A, I, or V; wherein H113 is substituted witheither a K, or R; wherein Q114 is substituted with a N; wherein D115 issubstituted with an E; wherein I116 is substituted with either an A, V,or L; wherein L117 is substituted with either an A, I, or V; whereinS118 is substituted with either an A, G, M, or T; wherein R119 issubstituted with either a K, or H; wherein V120 is substituted witheither an A, I, or L; wherein V121 is substituted with either an A, I,or L; wherein P122 is a P; wherein L123 is substituted with either an A,I, or V; wherein N124 is substituted with a Q; wherein Q125 issubstituted with a N; wherein S126 is substituted with either an A, G,M, or T; wherein F127 is substituted with either a W, or Y; wherein T128is substituted with either an A, G, M, or S; wherein E129 is substitutedwith a D; wherein K130 is substituted with either a R, or H; whereinY131 is either an F, or W; wherein A132 is substituted with either a G,I, L, M, S, T, or V; wherein G133 is substituted with either an A, M, S,or T; wherein I134 is substituted with either an A, V, or L; whereinF135 is substituted with either a W, or Y; wherein R136 is substitutedwith either a K, or H; wherein F137 is substituted with either a W, orY; wherein W138 is either an F, or Y; wherein F139 is substituted witheither a W, or Y; wherein W140 is either an F, or Y; wherein H141 issubstituted with either a K, or R; wherein Y142 is either an F, or W;wherein G143 is substituted with either an A, M, S, or T; wherein N144is substituted with a Q; wherein W145 is either an F, or Y; wherein V146is substituted with either an A, I, or L; wherein P147 is a P; whereinV148 is substituted with either an A, I, or L; wherein V149 issubstituted with either an A, I, or L; wherein I150 is substituted witheither an A, V, or L; wherein D151 is substituted with an E; whereinD152 is substituted with an E; wherein R153 is substituted with either aK, or H; wherein L154 is substituted with either an A, I, or V; whereinP155 is a P; wherein V156 is substituted with either an A, I, or L;wherein N157 is substituted with a Q; wherein E158 is substituted with aD; wherein A159 is substituted with either a G, I, L, M, S, T, or V;wherein G160 is substituted with either an A, M, S, or T; wherein Q161is substituted with a N; wherein L162 is substituted with either an A,I, or V; wherein V163 is substituted with either an A, I, or L; whereinF164 is substituted with either a W, or Y; wherein V165 is substitutedwith either an A, I, or L; wherein S166 is substituted with either an A,G, M, or T; wherein S167 is substituted with either an A, G, M, or T;wherein T168 is substituted with either an A, G, M, or S; wherein Y169is either an F, or W; wherein K170 is substituted with either a R, or H;wherein N171 is substituted with a Q; wherein L172 is substituted witheither an A, I, or V; wherein F173 is substituted with either a W, or Y;wherein W174 is either an F, or Y; wherein G175 is substituted witheither an A, M, S, or T; wherein A176 is substituted with either a G, I,L, M, S, T, or V; wherein L177 is substituted with either an A, I, or V;wherein L178 is substituted with either an A, I, or V; wherein E179 issubstituted with a D; wherein K180 is substituted with either a R, or H;wherein A181 is substituted with either a G, I, L, M, S, T, or V;wherein Y182 is either an F, or W; wherein A183 is substituted witheither a G, I, L, M, S, T, or V; wherein K184 is substituted with eithera R, or H; wherein L185 is substituted with either an A, I, or V;wherein S186 is substituted with either an A, G, M, or T; wherein G187is substituted with either an A, M, S, or T; wherein S188 is substitutedwith either an A, G, M, or T; wherein Y189 is either an F, or W; whereinE190 is substituted with a D; wherein D191 is substituted with an E;wherein L192 is substituted with either an A, I, or V; wherein Q193 issubstituted with a N; wherein S194 is substituted with either an A, G,M, or T; wherein G195 is substituted with either an A, M, S, or T;wherein Q196 is substituted with a N; wherein V197 is substituted witheither an A, I, or L; wherein S198 is substituted with either an A, G,M, or T; wherein E199 is substituted with a D; wherein A200 issubstituted with either a G, I, L, M, S, T, or V; wherein L201 issubstituted with either an A, I, or V; wherein V202 is substituted witheither an A, I, or L; wherein D203 is substituted with an E; whereinF204 is substituted with either a W, or Y; wherein T205 is substitutedwith either an A, G, M, or S; wherein G206 is substituted with either anA, M, S, or T; wherein G207 is substituted with either an A, M, S, or T;wherein V208 is substituted with either an A, I, or L; wherein T209 issubstituted with either an A, G, M, or S; wherein M210 is substitutedwith either an A, G, S, or T; wherein T211 is substituted with either anA, G, M, or S; wherein I212 is substituted with either an A, V, or L;wherein N213 is substituted with a Q; wherein L214 is substituted witheither an A, I, or V; wherein A215 is substituted with either a G, I, L,M, S, T, or V; wherein E216 is substituted with a D; wherein A217 issubstituted with either a G, I, L, M, S, T, or V; wherein H218 issubstituted with either a K, or R; wherein G219 is substituted witheither an A, M, S, or T; wherein N220 is substituted with a Q; whereinL221 is substituted with either an A, I, or V; wherein W222 is either anF, or Y; wherein D223 is substituted with an E; wherein I224 issubstituted with either an A, V, or L; wherein L225 is substituted witheither an A, I, or V; wherein I226 is substituted with either an A, V,or L; wherein E227 is substituted with a D; wherein A228 is substitutedwith either a G, I, L, M, S, T, or V; wherein T229 is substituted witheither an A, G, M, or S; wherein Y230 is either an F, or W; wherein N231is substituted with a Q; wherein R232 is substituted with either a K, orH; wherein T233 is substituted with either an A, G, M, or S; whereinL234 is substituted with either an A, I, or V; wherein I235 issubstituted with either an A, V, or L; wherein G236 is substituted witheither an A, M, S, or T; wherein C237 is a C; wherein Q238 issubstituted with a N; wherein T239 is substituted with either an A, G,M, or S; wherein H240 is substituted with either a K, or R; wherein S241is substituted with either an A, G, M, or T; wherein G242 is substitutedwith either an A, M, S, or T; wherein E243 is substituted with a D;wherein K244 is substituted with either a R, or H; wherein I245 issubstituted with either an A, V, or L; wherein L246 is substituted witheither an A, I, or V; wherein E247 is substituted with a D; wherein N248is substituted with a Q; wherein G249 is substituted with either an A,M, S, or T; wherein L250 is substituted with either an A, I, or V;wherein V251 is substituted with either an A, I, or L; wherein E252 issubstituted with a D; wherein G253 is substituted with either an A, M,S, or T; wherein H254 is substituted with either a K, or R; wherein A255is substituted with either a G, I, L, M, S, T, or V; wherein Y256 iseither an F, or W; wherein T257 is substituted with either an A, G, M,or S; wherein L258 is substituted with either an A, I, or V; whereinT259 is substituted with either an A, G, M, or S; wherein G260 issubstituted with either an A, M, S, or T; wherein I261 is substitutedwith either an A, V, or L; wherein R262 is substituted with either a K,or H; wherein K263 is substituted with either a R, or H; wherein V264 issubstituted with either an A, I, or L; wherein T265 is substituted witheither an A, G, M, or S; wherein C266 is a C; wherein K267 issubstituted with either a R, or H; wherein H268 is substituted witheither a K, or R; wherein R269 is substituted with either a K, or H;wherein P270 is a P; wherein E271 is substituted with a D; wherein Y272is either an F, or W; wherein L273 is substituted with either an A, I,or V; wherein V274 is substituted with either an A, I, or L; whereinK275 is substituted with either a R, or H; wherein L276 is substitutedwith either an A, I, or V; wherein R277 is substituted with either a K,or H; wherein N278 is substituted with a Q; wherein P279 is a P; whereinW280 is either an F, or Y; wherein G281 is substituted with either an A,M, S, or T; wherein K282 is substituted with either a R, or H; whereinV283 is substituted with either an A, I, or L; wherein E284 issubstituted with a D; wherein W285 is either an F, or Y; wherein K286 issubstituted with either a R, or H; wherein G287 is substituted witheither an A, M, S, or T; wherein D288 is substituted with an E; whereinW289 is either an F, or Y; wherein S290 is substituted with either an A,G, M, or T; wherein D291 is substituted with an E; wherein S292 issubstituted with either an A, G, M, or T; wherein S293 is substitutedwith either an A, G, M, or T; wherein S294 is substituted with either anA, G, M, or T; wherein K295 is substituted with either a R, or H;wherein W296 is either an F, or Y; wherein E297 is substituted with a D;wherein L298 is substituted with either an A, I, or V; wherein L299 issubstituted with either an A, I, or V; wherein S300 is substituted witheither an A, G, M, or T; wherein P301 is a P; wherein K302 issubstituted with either a R, or H; wherein E303 is substituted with a D;wherein K304 is substituted with either a R, or H; wherein I305 issubstituted with either an A, V, or L; wherein L306 is substituted witheither an A, I, or V; wherein L307 is substituted with either an A, I,or V; wherein L308 is substituted with either an A, I, or V; whereinR309 is substituted with either a K, or H; wherein K310 is substitutedwith either a R, or H; wherein D311 is substituted with an E; whereinN312 is substituted with a Q; wherein D313 is substituted with an E;wherein G314 is substituted with either an A, M, S, or T; wherein E315is substituted with a D; wherein F316 is substituted with either a W, orY; wherein W317 is either an F, or Y; wherein M318 is substituted witheither an A, G, S, or T; wherein T319 is substituted with either an A,G, M, or S; wherein L320 is substituted with either an A, I, or V;wherein Q321 is substituted with a N; wherein D322 is substituted withan E; wherein F323 is substituted with either a W, or Y; wherein K324 issubstituted with either a R, or H; wherein T325 is substituted witheither an A, G, M, or S; wherein H326 is substituted with either a K, orR; wherein F327 is substituted with either a W, or Y; wherein V328 issubstituted with either an A, I, or L; wherein L329 is substituted witheither an A, I, or V; wherein L330 is substituted with either an A, I,or V; and/or wherein V331 is substituted with either an A, I, or L ofSEQ ID NO:56 in addition to any combination thereof. Other suitablesubstitutions within the CAN-12v2 active site domain are encompassed bythe present invention and are referenced elsewhere herein. The presentinvention also encompasses the use of these CAN-12v2 active site domainconservative amino acid substituted polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

For purposes of the present invention, by “at least a portion of” ismeant all or any part of the CAN-12v2 active site domain defined by thestructure coordinates according to Table V (e.g., fragments thereof).More preferred are molecules comprising all or any parts of the CAN-12v2active site domain defined by the structure coordinates according toTable V, or a mutant or homologue of said molecule or molecular complex.By mutant or homologue of the molecule it is meant a molecule that has aroot mean square deviation from the backbone atoms of said CAN-12v2amino acids of not more than 4.5 Angstroms, and preferably not more than3.5 Angstroms.

The term “root mean square deviation” means the square root of thearithmetic mean of the squares of the deviations from the mean. It is aterm that expresses the deviation or variation from a trend or object.For the purposes of the present invention, the “root mean squaredeviation” defines the variation in the backbone of a protein from therelevant portion of the backbone of the AR portion of the complex asdefined by the structure coordinates described herein.

A preferred embodiment is a machine-readable data storage medium that iscapable of displaying a graphical three-dimensional representation of amolecule or molecular complex that is defined by the structurecoordinates of all of the amino acids in Table V±a root mean squaredeviation from the backbone atoms of those amino acids of not more than4.0 ANG, preferably 3.0 ANG.

The structure coordinates of a CAN-12v2 homology model, includingportions thereof, is stored in a machine-readable storage medium. Suchdata may be used for a variety of purposes, such as drug discovery.

Accordingly, in one embodiment of this invention is provided amachine-readable data storage medium comprising a data storage materialencoded with the structure coordinates set forth in Table V.

One embodiment utilizes System 10 as disclosed in WO 98/11134, thedisclosure of which is incorporated herein by reference in its entirety.Briefly, one version of these embodiments comprises a computercomprising a central processing unit (“CPU”), a working memory which maybe, e.g, RAM (random-access memory) or “core” memory, mass storagememory (such as one or more disk drives or CD-ROM drives), one or morecathode-ray tube (“CRT”) display terminals, one or more keyboards, oneor more input lines, and one or more output lines, all of which areinterconnected by a conventional bidirectional system bus.

Input hardware, coupled to the computer by input lines, may beimplemented in a variety of ways. Machine-readable data of thisinvention may be inputted via the use of a modem or modems connected bya telephone line or dedicated data line. Alternatively or additionally,the input hardware may comprise CD-ROM drives or disk drives. Inconjunction with a display terminal, keyboard may also be used as aninput device.

Output hardware, coupled to the computer by output lines, may similarlybe implemented by conventional devices. By way of example, outputhardware may include a CRT display terminal for displaying a graphicalrepresentation of a region or domain of the present invention using aprogram such as QUANTA as described herein. Output hardware might alsoinclude a printer, so that hard copy output may be produced, or a diskdrive, to store system output for later use.

In operation, the CPU coordinates the use of the various input andoutput devices, coordinates data accesses from mass storage, andaccesses to and from the working memory, and determines the sequence ofdata processing steps. A number of programs may be used to process themachine-readable data of this invention. Such programs are discussed inreference to the computational methods of drug discovery as describedherein. Specific references to components of the hardware system areincluded as appropriate throughout the following description of the datastorage medium.

For the purpose of the present invention, any magnetic data storagemedium which can be encoded with machine-readable data would besufficient for carrying out the storage requirements of the system. Themedium could be a conventional floppy diskette or hard disk, having asuitable substrate, which may be conventional, and a suitable coating,which may be conventional, on one or both sides, containing magneticdomains whose polarity or orientation could be altered magnetically, forexample. The medium may also have an opening for receiving the spindleof a disk drive or other data storage device.

The magnetic domains of the coating of a medium may be polarized ororiented so as to encode in a manner which may be conventional, machinereadable data such as that described herein, for execution by a systemsuch as the system described herein.

Another example of a suitable storage medium which could also be encodedwith such machine-readable data, or set of instructions, which could becarried out by a system such as the system described herein, could be anoptically-readable data storage medium. The medium could be aconventional compact disk read only memory (CD-ROM) or a rewritablemedium such as a magneto-optical disk which is optically readable andmagneto-optically writable. The medium preferably has a suitablesubstrate, which may be conventional, and a suitable coating, which maybe conventional, usually of one side of substrate.

In the case of a CD-ROM, as is well known, the coating is reflective andis impressed with a plurality of pits to encode the machine-readabledata. The arrangement of pits is read by reflecting laser light off thesurface of the coating. A protective coating, which preferably issubstantially transparent, is provided on top of the reflective coating.

In the case of a magneto-optical disk, as is well known, the coating hasno pits, but has a plurality of magnetic domains whose polarity ororientation can be changed magnetically when heated above a certaintemperature, as by a laser. The orientation of the domains can be readby measuring the polarization of laser light reflected from the coating.The arrangement of the domains encodes the data as described above.

Thus, in accordance with the present invention, data capable ofdisplaying the three dimensional structure of the CAN-12v2 homologymodel, or portions thereof and their structurally similar homologues isstored in a machine-readable storage medium, which is capable ofdisplaying a graphical three-dimensional representation of thestructure. Such data may be used for a variety of purposes, such as drugdiscovery.

For the first time, the present invention permits the use ofstructure-based or rational drug design techniques to design, select,and synthesize chemical entities that are capable of modulating thebiological function of CAN-12v2.

Accordingly, the present invention is also directed to the design ofsmall molecules which imitates the structure of the CAN-12v2 active sitedomain (SEQ ID NO:93), or a portion thereof defined by the structureprovided in Table V. Alternatively, the present invention is directed tothe design of small molecules which may bind to at least part of theCAN-12v2 active site domain (SEQ ID NO:93), or some portion thereof. Forpurposes of this invention, by CAN-12v2 active site domain, it is alsomeant to include mutants or homologues thereof. In a preferredembodiment, the mutants or homologues have at least 25% identity, morepreferably 50% identity, more preferably 75% identity, and mostpreferably 90% identity to SEQ ID NO:93. In this context, the term“small molecule” may be construed to mean any molecule described knownin the art or described elsewhere herein, though may include, forexample, peptides, chemicals, carbohydrates, nucleic acids, PNAs, andany derivatives thereof.

The three-dimensional model structure of CAN-12v2 will also providemethods for identifying modulators of biological function. Variousmethods or combination thereof can be used to identify these compounds.

For example, test compounds can be modeled that fit spatially into theactive site domain in CAN-12v2 embodied by the sequence from about R90to about amino acid C94, from about amino acid G99 to about amino acidL104, from about amino acid Q196 to about amino acid S198, from aboutamino acid M210 to about amino acid I212, from about amino acid G236 toabout amino acid H240, from about amino acid E252 to about amino acidY256, from about amino acid N278 to about amino acid K282, from aboutamino acid V328 to about amino acid V331, or some portion thereof, ofSEQ ID NO:56 (corresponding to SEQ ID NO:93), in accordance with thestructural coordinates of Table V.

Structure coordinates of the active site domain in CAN-12v2 defined bythe amino acids from about R90 to about amino acid C94, from about aminoacid G99 to about amino acid L104, from about amino acid Q196 to aboutamino acid S198, from about amino acid M210 to about amino acid I212,from about amino acid G236 to about amino acid H240, from about aminoacid E252 to about amino acid Y256, from about amino acid N278 to aboutamino acid K282, from about amino acid V328 to about amino acid V331 ofSEQ ID NO:56, can also be used to identify structural and chemicalfeatures. Identified structural or chemical features can then beemployed to design or select compounds as potential CAN-12v2 modulators.By structural and chemical features it is meant to include, but is notlimited to, van der Waals interactions, hydrogen bonding interactions,charge interaction, hydrophobic bonding interaction, and dipoleinteraction. Alternatively, or in conjunction with, thethree-dimensional structural model can be employed to design or selectcompounds as potential CAN-12v2 modulators. Compounds identified aspotential CAN-12v2 modulators can then be synthesized and screened in anassay characterized by binding of a test compound to the CAN-12v2, or incharacterizing the ability of CAN-12v2 to modulate a protease target inthe presence of a small molecule. Examples of assays useful in screeningof potential CAN-12v2 modulators include, but are not limited to,screening in silico, in vitro assays and high throughput assays.Finally, these methods may also involve modifying or replacing one ormore amino acids at amino acid positions, C101, H254, and/or N278 of SEQID NO:56 in accordance with the structure coordinates of Table V.

However, as will be understood by those of skill in the art upon thisdisclosure, other structure based design methods can be used. Variouscomputational structure based design methods have been disclosed in theart.

For example, a number of computer modeling systems are available inwhich the sequence of the CAN-12v2 and the CAN-12v2 structure (i.e.,atomic coordinates of CAN-12v2 and/or the atomic coordinates of theactive site domain as provided in Table V, can be input. This computersystem then generates the structural details of one or more theseregions in which a potential CAN-12v2 modulator binds so thatcomplementary structural details of the potential modulators can bedetermined. Design in these modeling systems is generally based upon thecompound being capable of physically and structurally associating withCAN-12v2. In addition, the compound must be able to assume aconformation that allows it to associate with CAN-12v2. Some modelingsystems estimate the potential inhibitory or binding effect of apotential CAN-12v2 modulator prior to actual synthesis and testing.

Methods for screening chemical entities or fragments for their abilityto associate with a given protein target are also well known. Oftenthese methods begin by visual inspection of the binding site on thecomputer screen. Selected fragments or chemical entities are thenpositioned in the active site domain of CAN-12v2. Docking isaccomplished using software such as INSIGHTII, QUANTA and SYBYL,following by energy minimization and molecular dynamics with standardmolecular mechanic forcefields such as CHARMM and AMBER. Examples ofcomputer programs which assist in the selection of chemical fragment orchemical entities useful in the present invention include, but are notlimited to, GRID (Goodford, 1985), AUTODOCK (Goodsell, 1990), and DOCK(Kuntz et al. 1982).

Upon selection of preferred chemical entities or fragments, theirrelationship to each other and CAN-12v2 can be visualized and thenassembled into a single potential modulator. Programs useful inassembling the individual chemical entities include, but are not limitedto CAVEAT (Bartlett et al. 1989) and 3D Database systems (Martin1992).

Alternatively, compounds may be designed de novo using either an emptyactive site or optionally including some portion of a known inhibitor.Methods of this type of design include, but are not limited to LUDI(Bohm 1992) and LeapFrog (Tripos Associates, St. Louis Mo.).

In addition, CAN-12v2 is overall well suited to modern methods includingcombinatorial chemistry.

Programs such as DOCK (Kuntz et al. 1982) can be used with the atomiccoordinates from the homology model to identify potential ligands fromdatabases or virtual databases which potentially bind CAN-12v2 activesite domain, and which may therefore be suitable candidates forsynthesis and testing.

Additionally, the three-dimensional homology model of CAN-12v2 will aidin the design of mutants with altered biological activity for theCAN-12v2 polypeptide.

The following are encompassed by the present invention: amachine-readable data storage medium, comprising a data storage materialencoded with machine readable data, wherein the data is defined by thestructure coordinates of the model CAN-12v2 according to Table V or ahomologue of said model, wherein said homologue comprises backbone atomsthat have a root mean square deviation from the backbone atoms of thecomplex of not more than 4.5 Å, preferably not more than 4.0 Å, mostpreferably not more than 3.5 Å, and even more preferably not more than3.0 Å; and a machine-readable data storage medium, wherein said moleculeis defined by the set of structure coordinates of the model for CAN-12v2according to Table V, or a homologue of said molecule, said homologuehaving a root mean square deviation from the backbone atoms of saidamino acids of not more than 4.5 Å, preferably not more than 4.0 Å, mostpreferably not more than 3.5 Å, and even more preferably not more than3.0 Å; a model comprising all or any part of the model defined bystructure coordinates of CAN-12v2 according to Table V, or a mutant orhomologue of said molecule or molecular complex.

In a further embodiment, the following are encompassed by the presentinvention: a method for identifying a mutant of CAN-12v2 with alteredbiological properties, function, or reactivity, the method comprisingany combination of steps of: use of the model or a homologue of saidmodel according to Table V, for the design of protein mutants withaltered biological function or properties which exhibit any combinationof therapeutic effects provided elsewhere herein; and use of the modelor a homologue of said model, for the design of a protein with mutationsin the active site domain comprised of the amino acids from about R90 toabout amino acid C94, from about amino acid G99 to about amino acidL104, from about amino acid Q196 to about amino acid S198, from aboutamino acid M210 to about amino acid I212, from about amino acid G236 toabout amino acid H240, from about amino acid E252 to about amino acidY256, from about amino acid N278 to about amino acid K282, from aboutamino acid V328 to about amino acid V331 of SEQ ID NO:56 according toTable V with altered biological function or properties which exhibit anycombination of therapeutic effects provided elsewhere herein.

In further preferred embodiments, the following are encompassed by thepresent invention: a method for identifying modulators of CAN-12v2biological properties, function, or reactivity, the method comprisingany combination of steps of: modeling test compounds that overlayspatially into the active site domain defined by all or any portion ofresidues from about R90 to about amino acid C94, from about amino acidG99 to about amino acid L104, from about amino acid Q196 to about aminoacid S198, from about amino acid M210 to about amino acid I212, fromabout amino acid G236 to about amino acid H240, from about amino acidE252 to about amino acid Y256, from about amino acid N278 to about aminoacid K282, from about amino acid V328 to about amino acid V331 of SEQ IDNO:56 according to Table V, or using a homologue or portion thereof.

The present invention encompasses using the structure coordinates as setforth herein to identify structural and chemical features of theCAN-12v2 polypeptide; employing identified structural or chemicalfeatures to design or select compounds as potential CAN-12v2 modulators;employing the three-dimensional structural model to design or selectcompounds as potential CAN-12v2 modulators; synthesizing the potentialCAN-12v2 modulators; screening the potential CAN-12v2 modulators in anassay characterized by binding of a protein to the CAN-12v2; selectingthe potential CAN-12v2 modulator from a database; designing the CAN-12v2modulator de novo; and/or designing said CAN-12v2 modulator from a knownmodulator activity.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO: 55 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 2090 ofSEQ ID NO:55, b is an integer between 15 to 2104, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ IDNO:55, and where b is greater than or equal to a+14.

In one embodiment, a CAN12.v2 polypeptide comprises a portion of theamino sequence depicted in FIGS. 9A-C. In another embodiment, a CAN12.v2polypeptide comprises at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 amino acids of the amino sequence depicted in FIGS.9A-C. In further embodiments, the CAN12.v2 polypeptide does not consistof the sequence ALLEKAYAKL (SEQ ID NO:141), and/or ALLEKAYAKLSGSYE. (SEQID NO:142) TABLE I ATCC NT Total 5′ NT 3′ AA Total Deposit SEQ NT ofStart NT Seq AA Gene CDNA No. Z ID. Seq of Codon of ID of No. CloneIDand Date Vector No. X Clone of ORF ORF No. Y ORF 1. CAN-12 XXXXX pSport1 1 and 4584 114 1397 24 428 (protease Xx/Xx/Xx 23 5, clone 70) 1.CAN-12 XXXXX pSport 1  1 4584 114 1995 2 581 (protease Xx/Xx/Xx 5, clone70, CAN- 12+) + splice amino acids 2. CAN- PTA- pSport 1 53 2095 9 209054 694 12v1 3434 (protease 06/07/01 5, clone 1e) 3. CAN- PTA- pSport 155 2104 9 2099 56 697 12v2 3434 (protease 06/07/01 5, clone 1e1b-1)

Table I summarizes the information corresponding to each “Gene No.”described above. The nucleotide sequence identified as “NT SEQ ID NO:X”was assembled from partially homologous (“overlapping”) sequencesobtained from the “cDNA clone ID” identified in Table I and, in somecases, from additional related DNA clones. The overlapping sequenceswere assembled into a single contiguous sequence of high redundancy(usually several overlapping sequences at each nucleotide position),resulting in a final sequence identified as SEQ ID NO:X.

The cDNA Clone ID was deposited on the date and given the correspondingdeposit number listed in “ATCC Deposit No:Z and Date.” “Vector” refersto the type of vector contained in the cDNA Clone ID.

“Total NT Seq. Of Clone” refers to the total number of nucleotides inthe clone contig identified by “Gene No.” The deposited clone maycontain all or most of the sequence of SEQ ID NO:X. The nucleotideposition of SEQ ID NO:X of the putative start codon (methionine) isidentified as “5′ NT of Start Codon of ORF.”

The translated amino acid sequence, beginning with the methionine, isidentified as “AA SEQ ID NO:Y,” although other reading frames can alsobe easily translated using known molecular biology techniques. Thepolypeptides produced by these alternative open reading frames arespecifically contemplated by the present invention.

The total number of amino acids within the open reading frame of SEQ IDNO:Y is identified as “Total AA of ORF”.

SEQ ID NO:X (where X may be any of the polynucleotide sequencesdisclosed in the sequence listing) and the translated SEQ ID NO:Y (whereY may be any of the polypeptide sequences disclosed in the sequencelisting) are sufficiently accurate and otherwise suitable for a varietyof uses well known in the art and described further herein. Forinstance, SEQ ID NO:X is useful for designing nucleic acid hybridizationprobes that will detect nucleic acid sequences contained in SEQ ID NO:Xor the cDNA contained in the deposited clone. These probes will alsohybridize to nucleic acid molecules in biological samples, therebyenabling a variety of forensic and diagnostic methods of the invention.Similarly, polypeptides identified from SEQ ID NO:Y may be used, forexample, to generate antibodies which bind specifically to proteinscontaining the polypeptides and the proteins encoded by the cDNA clonesidentified in Table I.

Nevertheless, DNA sequences generated by sequencing reactions cancontain sequencing errors. The errors exist as misidentifiednucleotides, or as insertions or deletions of nucleotides in thegenerated DNA sequence. The erroneously inserted or deleted nucleotidesmay cause frame shifts in the reading frames of the predicted amino acidsequence. In these cases, the predicted amino acid sequence divergesfrom the actual amino acid sequence, even though the generated DNAsequence may be greater than 99.9% identical to the actual DNA sequence(for example, one base insertion or deletion in an open reading frame ofover 1000 bases).

Accordingly, for those applications requiring precision in thenucleotide sequence or the amino acid sequence, the present inventionprovides not only the generated nucleotide sequence identified as SEQ IDNO:1, 23, 53, and/or 55 and the predicted translated amino acid sequenceidentified as SEQ ID NO:2, 24, 54, and/or 56, but also a sample ofplasmid DNA containing a cDNA of the invention deposited with the ATCC,as set forth in Table I. The nucleotide sequence of each deposited clonecan readily be determined by sequencing the deposited clone inaccordance with known methods. The predicted amino acid sequence canthen be verified from such deposits. Moreover, the amino acid sequenceof the protein encoded by a particular clone can also be directlydetermined by peptide sequencing or by expressing the protein in asuitable host cell containing the deposited cDNA, collecting theprotein, and determining its sequence.

The present invention also relates to the genes corresponding to SEQ IDNO:1, 23, 53, and/or 55, SEQ ID NO:2, 24, 54, and/or 56, or thedeposited clone. The corresponding gene can be isolated in accordancewith known methods using the sequence information disclosed herein. Suchmethods include preparing probes or primers from the disclosed sequenceand identifying or amplifying the corresponding gene from appropriatesources of genomic material.

Also provided in the present invention are species homologs, allelicvariants, and/or orthologs. The skilled artisan could, using procedureswell-known in the art, obtain the polynucleotide sequence correspondingto full-length genes (including, but not limited to the full-lengthcoding region), allelic variants, splice variants, orthologs, and/orspecies homologues of genes corresponding to SEQ ID NO:1, 23, 53, and/or55, SEQ ID NO:2, 24, 54, and/or 56, or a deposited clone, relying on thesequence from the sequences disclosed herein or the clones depositedwith the ATCC. For example, allelic variants and/or species homologuesmay be isolated and identified by making suitable probes or primerswhich correspond to the 5′, 3′, or internal regions of the sequencesprovided herein and screening a suitable nucleic acid source for allelicvariants and/or the desired homologue.

The polypeptides of the invention can be prepared in any suitablemanner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

The polypeptides may be in the form of the protein, or may be a part ofa larger protein, such as a fusion protein (see below). It is oftenadvantageous to include an additional amino acid sequence which containssecretory or leader sequences, pro-sequences, sequences which aid inpurification, such as multiple histidine residues, or an additionalsequence for stability during recombinant production.

The polypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Arecombinantly produced version of a polypeptide, can be substantiallypurified using techniques described herein or otherwise known in theart, such as, for example, by the one-step method described in Smith andJohnson, Gene 67:3140 (1988). Polypeptides of the invention also can bepurified from natural, synthetic or recombinant sources using protocolsdescribed herein or otherwise known in the art, such as, for example,antibodies of the invention raised against the full-length form of theprotein.

The present invention provides a polynucleotide comprising, oralternatively consisting of, the sequence identified as SEQ ID NO:1, 23,53, and/or 55, and/or a cDNA provided in ATCC Deposit No. Z:. Thepresent invention also provides a polypeptide comprising, oralternatively consisting of, the sequence identified as SEQ ID NO:2, 24,54, and/or 56, and/or a polypeptide encoded by the cDNA provided in ATCCDeposit NO:Z. The present invention also provides polynucleotidesencoding a polypeptide comprising, or alternatively consisting of thepolypeptide sequence of SEQ ID NO:2, 24, 54, and/or 56, and/or apolypeptide sequence encoded by the cDNA contained in ATCC Deposit No:Z.

Preferably, the present invention is directed to a polynucleotidecomprising, or alternatively consisting of, the sequence identified asSEQ ID NO:1, 23, 53, and/or 55, and/or a cDNA provided in ATCC DepositNo.: that is less than, or equal to, a polynucleotide sequence that is 5mega basepairs, 1 mega basepairs, 0.5 mega basepairs, 0.1 megabasepairs, 50,000 basepairs, 20,000 basepairs, or 10,000 basepairs inlength.

The present invention encompasses polynucleotides with sequencescomplementary to those of the polynucleotides of the present inventiondisclosed herein. Such sequences may be complementary to the sequencedisclosed as SEQ ID NO:1, 23, 53, and/or 55, the sequence contained in adeposit, and/or the nucleic acid sequence encoding the sequencedisclosed as SEQ ID NO:2, 24, 54, and/or 56.

The present invention also encompasses polynucleotides capable ofhybridizing, preferably under reduced stringency conditions, morepreferably under stringent conditions, and most preferably under highlystringent conditions, to polynucleotides described herein. Examples ofstringency conditions are shown in Table II below: highly stringentconditions are those that are at least as stringent as, for example,conditions A-F; stringent conditions are at least as stringent as, forexample, conditions G-L; and reduced stringency conditions are at leastas stringent as, for example, conditions M-R. TABLE II HybridizationWash Stringency Polynucleotide Hybrid Length Temperature and TemperatureCondition Hybrid± (bp)‡ Buffer† and Buffer† A DNA:DNA > or equal to 5065° C.; 1xSSC -or- 65° C.; 0.3xSSC 42° C.; 1xSSC, 50% formamide BDNA:DNA <50 Tb*; 1xSSC Tb*; 1xSSC C DNA:RNA > or equal to 50 67° C.;1xSSC -or- 67° C.; 0.3xSSC 45° C.; 1xSSC, 50% formamide D DNA:RNA <50Td*; 1xSSC Td*; 1xSSC E RNA:RNA > or equal to 50 70° C.; 1xSSC -or- 70°C.; 0.3xSSC 50° C.; 1xSSC, 50% formamide F RNA:RNA <50 Tf*; 1xSSC Tf*;1xSSC G DNA:DNA > or equal to 50 65° C.; 4xSSC -or- 65° C.; 1xSSC 45°C.; 4xSSC, 50% formamide H DNA:DNA <50 Th*; 4xSSC Th*; 4xSSC I DNA:RNA >or equal to 50 67° C.; 4xSSC -or- 67° C.; 1xSSC 45° C.; 4xSSC, 50%formamide J DNA:RNA <50 Tj*; 4xSSC Tj*; 4xSSC K RNA:RNA > or equal to 5070° C.; 4xSSC -or- 67° C.; 1xSSC 40° C.; 6xSSC, 50% formamide L RNA:RNA<50 Tl*; 2xSSC Tl*; 2xSSC M DNA:DNA > or equal to 50 50° C.; 4xSSC -or-50° C.; 2xSSC 40° C. 6xSSC, 50% formamide N DNA:DNA <50 Tn*; 6xSSC Tn*;6xSSC O DNA:RNA > or equal to 50 55° C.; 4xSSC -or- 55° C.; 2xSSC 42°C.; 6xSSC, 50% formamide P DNA:RNA <50 Tp*; 6xSSC Tp*; 6xSSC Q RNA:RNA >or equal to 50 60° C.; 4xSSC -or- 60° C.; 2xSSC 45° C.; 6xSSC, 50%formamide R RNA:RNA <50 Tr*; 4xSSC Tr*; 4xSSC‡The “hybrid length” is the anticipated length for the hybridizedregion(s) of the hybridizing polynucleotides. When hybridizing apolynucleotide of unknown sequence, the hybrid is assumed to be that ofthe hybridizing polynucleotide of the present invention. Whenpolynucleotides of known sequence are hybridized, the hybrid length canbe determined by aligning the sequences of the polynucleotides andidentifying the region or regions of optimal# sequence complementarity. Methods of aligning two or morepolynucleotide sequences and/or determining the percent identity betweentwo polynucleotide sequences are well known in the art (e.g., MegAlignprogram of the DNA*Star suite of programs, etc).†SSPE (1xSSPE is 0.15M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4)can be substituted for SSC (1xSSC is 0.15M NaCl and 15 mM sodiumcitrate) in the hybridization and wash buffers; washes are performed for15 minutes after hybridization is complete. The hydridizations andwashes may additionally include 5X Denhardt's reagent, .5-1.0% SDS, 100ug/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate,and up to 50% formamide.*Tb-Tr: The hybridization temperature for hybrids anticipated to be lessthan 50 base pairs in length should be 5-10° C. less than the meltingtemperature Tm of the hybrids there Tm is determined according to thefollowing equations. For hybrids less than 18 base pairs in length, Tm(°C.) = 2(# of A + T bases) + 4(# of G + C bases). For hybrids between 18# and 49 base pairs in length, Tm(° C.) = 81.5 + 16.6(log₁₀[Na+]) +0.41(% G + C) − (600/N), where N is the number of bases in the hybrid,and [Na+] is the concentration of sodium ions in the hybridizationbuffer ([NA+] for 1xSSC = .165 M).±The present invention encompasses the substitution of any one, or moreDNA or RNA hybrid partners with either a PNA, or a modifiedpolynucleotide. Such modified polynucleotides are known in the art andare more particularly described elsewhere herein.

Additional examples of stringency conditions for polynucleotidehybridization are provided, for example, in Sambrook, J., E. F. Fritsch,and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and11, and Current Protocols in Molecular Biology, 1995, F. M., Ausubel etal., eds, John Wiley and Sons, Inc., sections 2.10 and 6.3-6.4, whichare hereby incorporated by reference herein.

Preferably, such hybridizing polynucleotides have at least 70% sequenceidentity (more preferably, at least 80% identity; and most preferably atleast 90% or 95% identity) with the polynucleotide of the presentinvention to which they hybridize, where sequence identity is determinedby comparing the sequences of the hybridizing polynucleotides whenaligned so as to maximize overlap and identity while minimizing sequencegaps. The determination of identity is well known in the art, anddiscussed more specifically elsewhere herein.

The invention encompasses the application of PCR methodology to thepolynucleotide sequences of the present invention, the clone depositedwith the ATCC, and/or the cDNA encoding the polypeptides of the presentinvention. PCR techniques for the amplification of nucleic acids aredescribed in U.S. Pat. No. 4,683,195 and Saiki et al., Science,239:487-491 (1988). PCR, for example, may include the following steps,of denaturation of template nucleic acid (if double-stranded), annealingof primer to target, and polymerization. The nucleic acid probed or usedas a template in the amplification reaction may be genomic DNA, cDNA,RNA, or a PNA. PCR may be used to amplify specific sequences fromgenomic DNA, specific RNA sequence, and/or cDNA transcribed from mRNA.References for the general use of PCR techniques, including specificmethod parameters, include Mullis et al., Cold Spring Harbor Symp.Quant. Biol., 51:263, (1987), Ehrlich (ed), PCR Technology, StocktonPress, NY, 1989; Ehrlich et al., Science, 252:1643-1650, (1991); and“PCR Protocols, A Guide to Methods and Applications”, Eds., Innis etal., Academic Press, New York, (1990).

Signal Sequences

The present invention also encompasses mature forms of the polypeptidecomprising, or alternatively consisting of, the polypeptide sequence ofSEQ ID NO:2, 24, 54, and/or 56, the polypeptide encoded by thepolynucleotide described as SEQ ID NO:1, 23, 53, and/or 55, and/or thepolypeptide sequence encoded by a cDNA in the deposited clone. Thepresent invention also encompasses polynucleotides encoding mature formsof the present invention, such as, for example the polynucleotidesequence of SEQ ID NO:1, 23, 53, and/or 55, and/or the polynucleotidesequence provided in a cDNA of the deposited clone.

According to the signal hypothesis, proteins secreted by eukaryoticcells have a signal or secretary leader sequence which is cleaved fromthe mature protein once export of the growing protein chain across therough endoplasmic reticulum has been initiated. Most eukaryotic cellscleave secreted proteins with the same specificity. However, in somecases, cleavage of a secreted protein is not entirely uniform, whichresults in two or more mature species of the protein. Further, it haslong been known that cleavage specificity of a secreted protein isultimately determined by the primary structure of the complete protein,that is, it is inherent in the amino acid sequence of the polypeptide.

Methods for predicting whether a protein has a signal sequence, as wellas the cleavage point for that sequence, are available. For instance,the method of McGeoch, Virus Res. 3:271-286 (1985), uses the informationfrom a short N-terminal charged region and a subsequent uncharged regionof the complete (uncleaved) protein. The method of von Heinje, NucleicAcids Res. 14:4683-4690 (1986) uses the information from the residuessurrounding the cleavage site, typically residues −13 to +2, where +1indicates the amino terminus of the secreted protein. The accuracy ofpredicting the cleavage points of known mammalian secretory proteins foreach of these methods is in the range of 75-80%. (von Heinje, supra.)However, the two methods do not always produce the same predictedcleavage point(s) for a given protein.

The established method for identifying the location of signal sequences,in addition, to their cleavage sites has been the SignalP program (v1.1)developed by Henrik Nielsen et al., Protein Engineering 10:1-6 (1997).The program relies upon the algorithm developed by von Heinje, thoughprovides additional parameters to increase the prediction accuracy.

More recently, a hidden Markov model has been developed (H. Neilson, etal., Ismb 1998;6:122-30), which has been incorporated into the morerecent SignalP (v2.0). This new method increases the ability to identifythe cleavage site by discriminating between signal peptides anduncleaved signal anchors. The present invention encompasses theapplication of the method disclosed therein to the prediction of thesignal peptide location, including the cleavage site, to any of thepolypeptide sequences of the present invention.

As one of ordinary skill would appreciate, however, cleavage sitessometimes vary from organism to organism and cannot be predicted withabsolute certainty. Accordingly, the polypeptide of the presentinvention may contain a signal sequence. Polypeptides of the inventionwhich comprise a signal sequence have an N-terminus beginning within 5residues (i.e., + or −35 residues, or preferably at the −5, −4, −3, −2,−1, +1, +2, +3, +4, or 30 5 residue) of the predicted cleavage point.Similarly, it is also recognized that in some cases, cleavage of thesignal sequence from a secreted protein is not entirely uniform,resulting in more than one secreted species. These polypeptides, and thepolynucleotides encoding such polypeptides, are contemplated by thepresent invention.

Moreover, the signal sequence identified by the above analysis may notnecessarily predict the naturally occurring signal sequence. Forexample, the naturally occurring signal sequence may be further upstreamfrom the predicted signal sequence. However, it is likely that thepredicted signal sequence will be capable of directing the secretedprotein to the ER. Nonetheless, the present invention provides themature protein produced by expression of the polynucleotide sequence ofSEQ ID NO:1, 23, 53, and/or 55 and/or the polynucleotide sequencecontained in the cDNA of a deposited clone, in a mammalian cell (e.g.,COS cells, as described below). These polypeptides, and thepolynucleotides encoding such polypeptides, are contemplated by thepresent invention.

Polynucleotide and Polypeptide Variants

The present invention also encompasses variants (e.g., allelic variants,orthologs, etc.) of the polynucleotide sequence disclosed herein in SEQID NO:1, 23, 53, and/or 55, the complementary strand thereto, and/or thecDNA sequence contained in the deposited clone.

The present invention also encompasses variants of the polypeptidesequence, and/or fragments therein, disclosed in SEQ ID NO:2, 24, 54,and/or 56, a polypeptide encoded by the polynucleotide sequence in SEQID NO:1, 23, 53, and/or 55, and/or a polypeptide encoded by a cDNA inthe deposited clone.

“Variant” refers to a polynucleotide or polypeptide differing from thepolynucleotide or polypeptide of the present invention, but retainingessential properties thereof. Generally, variants are overall closelysimilar, and, in many regions, identical to the polynucleotide orpolypeptide of the present invention.

Thus, one aspect of the invention provides an isolated nucleic acidmolecule comprising, or alternatively consisting of, a polynucleotidehaving a nucleotide sequence selected from the group consisting of: (a)a nucleotide sequence encoding a CAN-12 related polypeptide having anamino acid sequence as shown in the sequence listing and described inSEQ ID NO:1, 23, 53, and/or 55 or the cDNA contained in ATCC depositNo:PTA-3434 ; (b) a nucleotide sequence encoding a mature CAN-12 relatedpolypeptide having the amino acid sequence as shown in the sequencelisting and described in SEQ ID NO:1, 23, 53, and/or 55 or the cDNAcontained in ATCC deposit No:PTA-3434 ; (c) a nucleotide sequenceencoding a biologically active fragment of a CAN-12 related polypeptidehaving an amino acid sequence shown in the sequence listing anddescribed in SEQ ID NO:1, 23, 53, and/or 55 or the cDNA contained inATCC deposit No:PTA-3434; (d) a nucleotide sequence encoding anantigenic fragment of a CAN-12 related polypeptide having an amino acidsequence sown in the sequence listing and described in SEQ ID NO:1, 23,53, and/or 55 or the cDNA contained in ATCC deposit No:PTA-3434; (e) anucleotide sequence encoding a CAN-12 related polypeptide comprising thecomplete amino acid sequence encoded by a human cDNA plasmid containedin SEQ ID NO:1, 23, 53, and/or 55 or the cDNA contained in ATCC depositNo:PTA-3434; (f) a nucleotide sequence encoding a mature CAN-12 relatedpolypeptide having an amino acid sequence encoded by a human cDNAplasmid contained in SEQ ID NO:1, 23, 53, and/or 55 or the cDNAcontained in ATCC deposit No:PTA-3434; (g) a nucleotide sequenceencoding a biologically active fragment of a CAN-12 related polypeptidehaving an amino acid sequence encoded by a human cDNA plasmid containedin SEQ ID NO:1, 23, 53, and/or 55 or the cDNA contained in ATCC depositNo:PTA-3434; (h) a nucleotide sequence encoding an antigenic fragment ofa CAN-12 related polypeptide having an amino acid sequence encoded by ahuman cDNA plasmid contained in SEQ ID NO:1, 23, 53, and/or 55 or thecDNA contained in ATCC deposit No:PTA-3434; (I) a nucleotide sequencecomplimentary to any of the nucleotide sequences in (a), (b), (c), (d),(e), (f), (g), or (h), above.

The present invention is also directed to polynucleotide sequences whichcomprise, or alternatively consist of, a polynucleotide sequence whichis at least about 80%, 85%, 90%, 91%, 92%, 92.4%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,or 99.9% identical to, for example, any of the nucleotide sequences in(a), (b), (c), (d), (e), (f), (g), or (h), above. Polynucleotidesencoded by these nucleic acid molecules are also encompassed by theinvention. In another embodiment, the invention encompasses nucleic acidmolecules which comprise, or alternatively, consist of a polynucleotidewhich hybridizes under stringent conditions, or alternatively, underlower stringency conditions, to a polynucleotide in (a), (b), (c), (d),(e), (f), (g), or (h), above. Polynucleotides which hybridize to thecomplement of these nucleic acid molecules under stringent hybridizationconditions or alternatively, under lower stringency conditions, are alsoencompassed by the invention, as are polypeptides encoded by thesepolypeptides.

Another aspect of the invention provides an isolated nucleic acidmolecule comprising, or alternatively, consisting of, a polynucleotidehaving a nucleotide sequence selected from the group consisting of: (a)a nucleotide sequence encoding a CAN-12 related polypeptide having anamino acid sequence as shown in the sequence listing and descried inTable I; (b) a nucleotide sequence encoding a mature CAN-12 relatedpolypeptide having the amino acid sequence as shown in the sequencelisting and descried in Table I; (c) a nucleotide sequence encoding abiologically active fragment of a CAN-12 related polypeptide having anamino acid sequence as shown in the sequence listing and descried inTable I; (d) a nucleotide sequence encoding an antigenic fragment of aCAN-12 related polypeptide having an amino acid sequence as shown in thesequence listing and descried in Table I; (e) a nucleotide sequenceencoding a CAN-12 related polypeptide comprising the complete amino acidsequence encoded by a human cDNA in a cDNA plasmid contained in the ATCCDeposit and described in Table I; (f) a nucleotide sequence encoding amature CAN-12 related polypeptide having an amino acid sequence encodedby a human cDNA in a cDNA plasmid contained in the ATCC Deposit anddescribed in Table I: (g) a nucleotide sequence encoding a biologicallyactive fragment of a CAN-12 related polypeptide having an amino acidsequence encoded by a human cDNA in a cDNA plasmid contained in the ATCCDeposit and described in Table I; (h) a nucleotide sequence encoding anantigenic fragment of a CAN-12 related polypeptide having an amino acidsequence encoded by a human cDNA in a cDNA plasmid contained in the ATCCdeposit and described in Table I; (i) a nucleotide sequencecomplimentary to any of the nucleotide sequences in (a), (b), (c), (d),(e), (f), (g), or (h) above.

The present invention is also directed to nucleic acid molecules whichcomprise, or alternatively, consist of, a nucleotide sequence which isat least about 80%, 85%, 90%, 91%, 92%, 92.4%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or99.9% identical to, for example, any of the nucleotide sequences in (a),(b), (c), (d), (e), (f), (g), or (h), above.

The present invention encompasses polypeptide sequences which comprise,or alternatively consist of, an amino acid sequence which is at leastabout 60.3%, 61.4%, 80%, 81.8%, 82.2%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8%, or 99.9% identical to, the following non-limited examples,the polypeptide sequence identified as SEQ ID NO:2, 24, 54, and/or 56,the polypeptide sequence encoded by a cDNA provided in the depositedclone, and/or polypeptide fragments of any of the polypeptides providedherein. Polynucleotides encoded by these nucleic acid molecules are alsoencompassed by the invention. In another embodiment, the inventionencompasses nucleic acid molecules which comprise, or alternatively,consist of a polynucleotide which hybridizes under stringent conditions,or alternatively, under lower stringency conditions, to a polynucleotidein (a), (b), (c), (d), (e), (f), (g), or (h), above. Polynucleotideswhich hybridize to the complement of these nucleic acid molecules understringent hybridization conditions or alternatively, under lowerstringency conditions, are also encompassed by the invention, as arepolypeptides encoded by these polypeptides.

The present invention is also directed to polypeptides which comprise,or alternatively consist of, an amino acid sequence which is at leastabout 60.3%, 61.4%, 80%, 81.8%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.1%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or99.9% identical to, for example, the polypeptide sequence shown in SEQID NO:2, 24, 54, and/or 56, a polypeptide sequence encoded by thenucleotide sequence in SEQ ID NO:1, 23, 53, and/or 55, a polypeptidesequence encoded by the cDNA in cDNA plasmid:Z, and/or polypeptidefragments of any of these polypeptides (e.g., those fragments describedherein). Polynucleotides which hybridize to the complement of thenucleic acid molecules encoding these polypeptides under stringenthybridization conditions or alternatively, under lower stringencyconditions, are also encompasses by the present invention, as are thepolypeptides encoded by these polynucleotides.

By a nucleic acid having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence of the presentinvention, it is intended that the nucleotide sequence of the nucleicacid is identical to the reference sequence except that the nucleotidesequence may include up to five point mutations per each 100 nucleotidesof the reference nucleotide sequence encoding the polypeptide. In otherwords, to obtain a nucleic acid having a nucleotide sequence at least95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. The query sequence may be an entire sequence referenced inTable I, the ORF (open reading frame), or any fragment specified asdescribed herein.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least about 60.3%, 61.4%, 80%, 81.8%, 80%, 85%, 90%,91%, 92%, 92.4%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a nucleotidesequence of the present invention can be determined conventionally usingknown computer programs. A preferred method for determining the bestoverall match between a query sequence (a sequence of the presentinvention) and a subject sequence, also referred to as a global sequencealignment, can be determined using the CLUSTALW computer program(Thompson, J. D., et al., Nucleic Acids Research, 2(22):4673-4680,(1994)), which is based on the algorithm of Higgins, D. G., et al.,Computer Applications in the Biosciences (CABIOS), 8(2):189-191, (1992).In a sequence alignment the query and subject sequences are both DNAsequences. An RNA sequence can be compared by converting U's to T's.However, the CLUSTALW algorithm automatically converts U's to T's whencomparing RNA sequences to DNA sequences. The result of said globalsequence alignment is in percent identity. Preferred parameters used ina CLUSTALW alignment of DNA sequences to calculate percent identity viapairwise alignments are: Matrix=IUB, k-tuple=1, Number of TopDiagonals=5, Gap Penalty=3, Gap Open Penalty 10, Gap ExtensionPenalty=0.1, Scoring Method=Percent, Window Size=5 or the length of thesubject nucleotide sequence, whichever is shorter. For multiplealignments, the following CLUSTALW parameters are preferred: Gap OpeningPenalty=10; Gap Extension Parameter=0.05; Gap Separation PenaltyRange=8; End Gap Separation Penalty=Off; % Identity for AlignmentDelay=40%; Residue Specific Gaps:Off; Hydrophilic Residue Gap=Off; andTransition Weighting=0. The pairwise and multple alignment parametersprovided for CLUSTALW above represent the default parameters as providedwith the AlignX software program (Vector NTI suite of programs, version6.0).

The present invention encompasses the application of a manual correctionto the percent identity results, in the instance where the subjectsequence is shorter than the query sequence because of 5′ or 3′deletions, not because of internal deletions. If only the local pairwisepercent identity is required, no manual correction is needed. However, amanual correction may be applied to determine the global percentidentity from a global polynucleotide alignment. Percent identitycalculations based upon global polynucleotide alignments are oftenpreferred since they reflect the percent identity between thepolynucleotide molecules as a whole (i.e., including any polynucleotideoverhangs, not just overlapping regions), as opposed to, only localmatching polynucleotides. Manual corrections for global percent identitydeterminations are required since the CLUSTALW program does not accountfor 5′ and 3′ truncations of the subject sequence when calculatingpercent identity. For subject sequences truncated at the 5′ or 3′ ends,relative to the query sequence, the percent identity is corrected bycalculating the number of bases of the query sequence that are 5′ and 3′of the subject sequence, which are not matched/aligned, as a percent ofthe total bases of the query sequence. Whether a nucleotide ismatched/aligned is determined by results of the CLUSTALW sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above CLUSTALW program using the specified parameters,to arrive at a final percent identity score. This corrected score may beused for the purposes of the present invention. Only bases outside the5′ and 3′ bases of the subject sequence, as displayed by the CLUSTALWalignment, which are not matched/aligned with the query sequence, arecalculated for the purposes of manually adjusting the percent identityscore.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the CLUSTALW alignment doesnot show a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theCLUSTALW program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by CLUSTALW is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are required for thepurposes of the present invention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% of the amino acid residues in thesubject sequence may be inserted, deleted, or substituted with anotheramino acid. These alterations of the reference sequence may occur at theamino- or carboxy-terminal positions of the reference amino acidsequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at leastabout 60.3%, 61.4%, 80%, 81.8%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8%, or 99.9% identical to, for instance, an amino acid sequencereferenced in Table 1 (SEQ ID NO:2) or to the amino acid sequenceencoded by cDNA contained in a deposited clone, can be determinedconventionally using known computer programs. A preferred method fordetermining the best overall match between a query sequence (a sequenceof the present invention) and a subject sequence, also referred to as aglobal sequence alignment, can be determined using the CLUSTALW computerprogram (Thompson, J. D., et al., Nucleic Acids Research,2(22):4673-4680, (1994)), which is based on the algorithm of Higgins, D.G., et al., Computer Applications in the Biosciences (CABIOS),8(2):189-191, (1992). In a sequence alignment the query and subjectsequences are both amino acid sequences. The result of said globalsequence alignment is in percent identity. Preferred parameters used ina CLUSTALW alignment of DNA sequences to calculate percent identity viapairwise alignments are: Matrix=BLOSUM, k-tuple=1, Number of TopDiagonals=5, Gap Penalty=3, Gap Open Penalty 10, Gap ExtensionPenalty=0.1, Scoring Method=Percent, Window Size=5 or the length of thesubject nucleotide sequence, whichever is shorter. For multiplealignments, the following CLUSTALW parameters are preferred: Gap OpeningPenalty=10; Gap Extension Parameter=0.05; Gap Separation PenaltyRange=8; End Gap Separation Penalty=Off; % Identity for AlignmentDelay=40%; Residue Specific Gaps:Off; Hydrophilic Residue Gap=Off; andTransition Weighting=0. The pairwise and multple alignment parametersprovided for CLUSTALW above represent the default parameters as providedwith the AlignX software program (Vector NTI suite of programs, version6.0).

The present invention encompasses the application of a manual correctionto the percent identity results, in the instance where the subjectsequence is shorter than the query sequence because of N— or C-terminaldeletions, not because of internal deletions. If only the local pairwisepercent identity is required, no manual correction is needed. However, amanual correction may be applied to determine the global percentidentity from a global polypeptide alignment. Percent identitycalculations based upon global polypeptide alignments are oftenpreferred since they reflect the percent identity between thepolypeptide molecules as a whole (i.e., including any polypeptideoverhangs, not just overlapping regions), as opposed to, only localmatching polypeptides. Manual corrections for global percent identitydeterminations are required since the CLUSTALW program does not accountfor N— and C-terminal truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the N—and C-termini, relative to the query sequence, the percent identity iscorrected by calculating the number of residues of the query sequencethat are N— and C-terminal of the subject sequence, which are notmatched/aligned with a corresponding subject residue, as a percent ofthe total bases of the query sequence. Whether a residue ismatched/aligned is determined by results of the CLUSTALW sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above CLUSTALW program using the specified parameters,to arrive at a final percent identity score. This final percent identityscore is what may be used for the purposes of the present invention.Only residues to the N— and C-termini of the subject sequence, which arenot matched/aligned with the query sequence, are considered for thepurposes of manually adjusting the percent identity score. That is, onlyquery residue positions outside the farthest N— and C-terminal residuesof the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theCLUSTALW alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N— and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the CLUSTALWprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N— orC-termini of the subject sequence, which are not matched/aligned withthe query. In this case the percent identity calculated by CLUSTALW isnot manually corrected. Once again, only residue positions outside theN— and C-terminal ends of the subject sequence, as displayed in theCLUSTALW alignment, which are not matched/aligned with the querysequence are manually corrected for. No other manual corrections arerequired for the purposes of the present invention.

In addition to the above method of aligning two or more polynucleotideor polypeptide sequences to arrive at a percent identity value for thealigned sequences, it may be desirable in some circumstances to use amodified version of the CLUSTALW algorithm which takes into accountknown structural features of the sequences to be aligned, such as forexample, the SWISS-PROT designations for each sequence. The result ofsuch a modifed CLUSTALW algorithm may provide a more accurate value ofthe percent identity for two polynucleotide or polypeptide sequences.Support for such a modified version of CLUSTALW is provided within theCLUSTALW algorithm and would be readily appreciated to one of skill inthe art of bioinformatics.

The variants may contain alterations in the coding regions, non-codingregions, or both. Especially preferred are polynucleotide variantscontaining alterations which produce silent substitutions, additions, ordeletions, but do not alter the properties or activities of the encodedpolypeptide. Nucleotide variants produced by silent substitutions due tothe degeneracy of the genetic code are preferred. Moreover, variants inwhich 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or addedin any combination are also preferred. Polynucleotide variants can beproduced for a variety of reasons, e.g., to optimize codon expressionfor a particular host (change codons in the MRNA to those preferred by abacterial host such as E. coli).

Naturally occurring variants are called “allelic variants,” and refer toone of several alternate forms of a gene occupying a given locus on achromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985).) These allelic variants can vary at either thepolynucleotide and/or polypeptide level and are included in the presentinvention. Alternatively, non-naturally occurring variants may beproduced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the polypeptides of the present invention. Forinstance, one or more amino acids can be deleted from the N-terminus orC-terminus of the protein without substantial loss of biologicalfunction. The authors of Ron et al., J. Biol. Chem. 268: 2984-2988(1993), reported variant KGF proteins having heparin binding activityeven after deleting 3, 8, or 27 amino-terminal amino acid residues.Similarly, Interferon gamma exhibited up to ten times higher activityafter deleting 8-10 amino acid residues from the carboxy terminus ofthis protein (Dobeli et al., J. Biotechnology 7:199-216 (1988)).

Moreover, ample evidence demonstrates that variants often retain abiological activity similar to that of the naturally occurring protein.For example, Gayle and coworkers (J. Biol. Chem. 268:22105-22111 (1993))conducted extensive mutational analysis of human cytokine IL-1a. Theyused random mutagenesis to generate over 3,500 individual IL-1a mutantsthat averaged 2.5 amino acid changes per variant over the entire lengthof the molecule. Multiple mutations were examined at every possibleamino acid position. The investigators found that “[m]ost of themolecule could be altered with little effect on either [binding orbiological activity].” In fact, only 23 unique amino acid sequences, outof more than 3,500 nucleotide sequences examined, produced a proteinthat significantly differed in activity from wild-type.

Furthermore, even if deleting one or more amino acids from theN-terminus or C-terminus of a polypeptide results in modification orloss of one or more biological functions, other biological activitiesmay still be retained. For example, the ability of a deletion variant toinduce and/or to bind antibodies which recognize the protein will likelybe retained when less than the majority of the residues of the proteinare removed from the N-terminus or C-terminus. Whether a particularpolypeptide lacking N— or C-terminal residues of a protein retains suchimmunogenic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art.

Alternatively, such N-terminus or C-terminus deletions of a polypeptideof the present invention may, in fact, result in a significant increasein one or more of the biological activities of the polypeptide(s). Forexample, biological activity of many polypeptides are governed by thepresence of regulatory domains at either one or both termini. Suchregulatory domains effectively inhibit the biological activity of suchpolypeptides in lieu of an activation event (e.g., binding to a cognateligand or receptor, phosphorylation, proteolytic processing, etc.).Thus, by eliminating the regulatory domain of a polypeptide, thepolypeptide may effectively be rendered biologically active in theabsence of an activation event.

Thus, the invention further includes polypeptide variants that showsubstantial biological activity. Such variants include deletions,insertions, inversions, repeats, and substitutions selected according togeneral rules known in the art so as have little effect on activity. Forexample, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie et al., Science 247:1306-1310(1990), wherein the authors indicate that there are two main strategiesfor studying the tolerance of an amino acid sequence to change.

The first strategy exploits the tolerance of amino acid substitutions bynatural selection during the process of evolution. By comparing aminoacid sequences in different species, conserved amino acids can beidentified. These conserved amino acids are likely important for proteinfunction. In contrast, the amino acid positions where substitutions havebeen tolerated by natural selection indicates that these positions arenot critical for protein function. Thus, positions tolerating amino acidsubstitution could be modified while still maintaining biologicalactivity of the protein.

The second strategy uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. (Cunningham and Wells,Science 244:1081-1085 (1989).) The resulting mutant molecules can thenbe tested for biological activity.

As the authors state, these two strategies have revealed that proteinsare surprisingly tolerant of amino acid substitutions. The authorsfurther indicate which amino acid changes are likely to be permissive atcertain amino acid positions in the protein. For example, most buried(within the tertiary structure of the protein) amino acid residuesrequire nonpolar side chains, whereas few features of surface sidechains are generally conserved.

The invention encompasses polypeptides having a lower degree of identitybut having sufficient similarity so as to perform one or more of thesame functions performed by the polypeptide of the present invention.Similarity is determined by conserved amino acid substitution. Suchsubstitutions are those that substitute a given amino acid in apolypeptide by another amino acid of like characteristics (e.g.,chemical properties). According to Cunningham et al above, suchconservative substitutions are likely to be phenotypically silent.Additional guidance concerning which amino acid changes are likely to bephenotypically silent are found in Bowie et al., Science 247:1306-1310(1990).

Tolerated conservative amino acid substitutions of the present inventioninvolve replacement of the aliphatic or hydrophobic amino acids Ala,Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr;replacement of the acidic residues Asp and Glu; replacement of the amideresidues Asn and Gln, replacement of the basic residues Lys, Arg, andHis; replacement of the aromatic residues Phe, Tyr, and Trp, andreplacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

In addition, the present invention also encompasses the conservativesubstitutions provided in Table III below. TABLE III For Amino Acid CodeReplace with any of: Alanine A D-Ala, Gly, beta-Ala, L-Cys, D-CysArginine R D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile, D-Met, D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-GlnAspartic Acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine CD-Cys, S—Me-Cys, Met, D-Met, Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asn,Glu, D-Glu, Asp, D-Asp Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn,Gln, D-Gln Glycine G Ala, D-Ala, Pro, D-Pro, β-Ala, Acp Isoleucine ID-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu, Val, D-Val,Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg, Met, D-Met,Ile, D- Ile, Orn, D-Orn Methionine M D-Met, S—Me-Cys, Ile, D-Ile, Leu,D-Leu, Val, D-Val Phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa, His, D-His,Trp, D-Trp, Trans-3,4, or 5-phenylproline, cis-3,4, or 5-phenylprolineProline P D-Pro, L-1-thioazolidine-4-carboxylic acid, D- or L-1-oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr, allo-Thr, Met,D-Met, Met(O), D-Met(O), L- Cys, D-Cys Threonine T D-Thr, Ser, D-Ser,allo-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-Val Tyrosine Y D-Tyr,Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Ile, D-Ile,Met, D-Met

Aside from the uses described above, such amino acid substitutions mayalso increase protein or peptide stability. The invention encompassesamino acid substitutions that contain, for example, one or morenon-peptide bonds (which replace the peptide bonds) in the protein orpeptide sequence. Also included are substitutions that include aminoacid residues other than naturally occurring L-amino acids, e.g.,D-amino acids or non-naturally occurring or synthetic amino acids, e.g.,B or y amino acids.

Both identity and similarity can be readily calculated by reference tothe following publications: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Informatics Computer Analysis of Sequence Data, Part 1,Griffin, A. M., and Griffin, H. G., eds., Humana Press,New Jersey, 1994;Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press,1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds.,M Stockton Press, New York, 1991.

In addition, the present invention also encompasses substitution ofamino acids based upon the probability of an amino acid substitutionresulting in conservation of function. Such probabilities are determinedby aligning multiple genes with related function and assessing therelative penalty of each substitution to proper gene function. Suchprobabilities are often described in a matrix and are used by somealgorithms (e.g., BLAST, CLUSTALW, GAP, etc.) in calculating percentsimilarity wherein similarity refers to the degree by which one aminoacid may substitute for another amino acid without lose of function. Anexample of such a matrix is the PAM250 or BLOSUM62 matrix.

Aside from the canonical chemically conservative substitutionsreferenced above, the invention also encompasses substitutions which aretypically not classified as conservative, but that may be chemicallyconservative under certain circumstances. Analysis of enzymaticcatalysis for proteases, for example, has shown that certain amino acidswithin the active site of some enzymes may have highly perturbed pKa'sdue to the unique microenvironment of the active site. Such perturbedpKa's could enable some amino acids to substitute for other amino acidswhile conserving enzymatic structure and function. Examples of aminoacids that are known to have amino acids with perturbed pKa's are theGlu-35 residue of Lysozyme, the Ile-16 residue of Chymotrypsin, theHis-159 residue of Papain, etc. The conservation of function relates toeither anomalous protonation or anomalous deprotonation of such aminoacids, relative to their canonical, non-perturbed pKa. The pKaperturbation may enable these amino acids to actively participate ingeneral acid-base catalysis due to the unique ionization environmentwithin the enzyme active site. Thus, substituting an amino acid capableof serving as either a general acid or general base within themicroenvironment of an enzyme active site or cavity, as may be the case,in the same or similar capacity as the wild-type amino acid, wouldeffectively serve as a conservative amino substitution.

Besides conservative amino acid substitution, variants of the presentinvention include, but are not limited to, the following: (i)substitutions with one or more of the non-conserved amino acid residues,where the substituted amino acid residues may or may not be one encodedby the genetic code, or (ii) substitution with one or more of amino acidresidues having a substituent group, or (iii) fusion of the maturepolypeptide with another compound, such as a compound to increase thestability and/or solubility of the polypeptide (for example,polyethylene glycol), or (iv) fusion of the polypeptide with additionalamino acids, such as, for example, an IgG Fc fusion region peptide, orleader or secretory sequence, or a sequence facilitating purification.Such variant polypeptides are deemed to be within the scope of thoseskilled in the art from the teachings herein.

For example, polypeptide variants containing amino acid substitutions ofcharged amino acids with other charged or neutral amino acids mayproduce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).)

Moreover, the invention further includes polypeptide variants createdthrough the application of molecular evolution (“DNA Shuffling”)methodology to the polynucleotide disclosed as SEQ ID NO:1, 23, 53,and/or 55, the sequence of the clone submitted in a deposit, and/or thecDNA encoding the polypeptide disclosed as SEQ ID NO:2, 24, 54, and/or56. Such DNA Shuffling technology is known in the art and moreparticularly described elsewhere herein (e.g., WPC, Stemmer, PNAS,91:10747, (1994)), and in the Examples provided herein).

A further embodiment of the invention relates to a polypeptide whichcomprises the amino acid sequence of the present invention having anamino acid sequence which contains at least one amino acid substitution,but not more than 50 amino acid substitutions, even more preferably, notmore than 40 amino acid substitutions, still more preferably, not morethan 30 amino acid substitutions, and still even more preferably, notmore than 20 amino acid substitutions. Of course, in order ofever-increasing preference, it is highly preferable for a peptide orpolypeptide to have an amino acid sequence which comprises the aminoacid sequence of the present invention, which contains at least one, butnot more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions.In specific embodiments, the number of additions, substitutions, and/ordeletions in the amino acid sequence of the present invention orfragments thereof (e.g., the mature form and/or other fragmentsdescribed herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150,conservative amino acid substitutions are preferable.

Polynucleotide and Polypeptide Fragments

The present invention is directed to polynucleotide fragments of thepolynucleotides of the invention, in addition to polypeptides encodedtherein by said polynucleotides and/or fragments.

In the present invention, a “polynucleotide fragment” refers to a shortpolynucleotide having a nucleic acid sequence which: is a portion ofthat contained in a deposited clone, or encoding the polypeptide encodedby the cDNA in a deposited clone; is a portion of that shown in SEQ IDNO:1, 23, 53, and/or 55 or the complementary strand thereto, or is aportion of a polynucleotide sequence encoding the polypeptide of SEQ IDNO:2, 24, 54, and/or 56. The nucleotide fragments of the invention arepreferably at least about 15 nt, and more preferably at least about 20nt, still more preferably at least about 30 nt, and even morepreferably, at least about 40 nt, at least about 50 nt, at least about75 nt, or at least about 150 nt in length. A fragment “at least 20 nt inlength,” for example, is intended to include 20 or more contiguous basesfrom the cDNA sequence contained in a deposited clone or the nucleotidesequence shown in SEQ ID NO:1, 23, 53, and/or 55. In this context“about” includes the particularly recited value, a value larger orsmaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus,or at both termini. These nucleotide fragments have uses that include,but are not limited to, as diagnostic probes and primers as discussedherein. Of course, larger fragments (e.g., 50, 150, 500, 600, 2000nucleotides) are preferred.

Moreover, representative examples of polynucleotide fragments of theinvention, include, for example, fragments comprising, or alternativelyconsisting of, a sequence from about nucleotide number 1-50, 51-100,101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500,501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950,951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250,1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550,1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850,1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ ID NO:1, 23,53, and/or 55, or the complementary strand thereto, or the cDNAcontained in a deposited clone. In this context “about” includes theparticularly recited ranges, and ranges larger or smaller by several (5,4, 3, 2, or 1) nucleotides, at either terminus or at both termini.Preferably, these fragments encode a polypeptide which has biologicalactivity. More preferably, these polynucleotides can be used as probesor primers as discussed herein. Also encompassed by the presentinvention are polynucleotides which hybridize to these nucleic acidmolecules under stringent hybridization conditions or lower stringencyconditions, as are the polypeptides encoded by these polynucleotides.

In the present invention, a “polypeptide fragment” refers to an aminoacid sequence which is a portion of that contained in SEQ ID NO:2, 24,54, and/or 56 or encoded by the cDNA contained in a deposited clone.Protein (polypeptide) fragments may be “free-standing,” or comprisedwithin a larger polypeptide of which the fragment forms a part orregion, most preferably as a single continuous region. Representativeexamples of polypeptide fragments of the invention, include, forexample, fragments comprising, or alternatively consisting of, fromabout amino acid number 1-20, 21-40, 41-60, 61-80, 81 - 100, 102-120,121-140, 141-160, or 161 to the end of the coding region. Moreover,polypeptide fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, or 150 amino acids in length. In this context“about” includes the particularly recited ranges or values, and rangesor values larger or smaller by several (5, 4, 3, 2, or 1) amino acids,at either extreme or at both extremes. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

Preferred polypeptide fragments include the full-length protein. Furtherpreferred polypeptide fragments include the full-length protein having acontinuous series of deleted residues from the amino or the carboxyterminus, or both. For example, any number of amino acids, ranging from1-60, can be deleted from the amino terminus of the full-lengthpolypeptide. Similarly, any number of amino acids, ranging from 1-30,can be deleted from the carboxy terminus of the full-length protein.Furthermore, any combination of the above amino and carboxy terminusdeletions are preferred. Similarly, polynucleotides encoding thesepolypeptide fragments are also preferred.

Also preferred are polypeptide and polynucleotide fragmentscharacterized by structural or functional domains, such as fragmentsthat comprise alpha-helix and alpha-helix forming regions, beta-sheetand beta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions. Polypeptide fragments of SEQ ID NO:2, 24, 54, and/or 56falling within conserved domains are specifically contemplated by thepresent invention. Moreover, polynucleotides encoding these domains arealso contemplated.

Other preferred polypeptide fragments are biologically active fragments.Biologically active fragments are those exhibiting activity similar, butnot necessarily identical, to an activity of the polypeptide of thepresent invention. The biological activity of the fragments may includean improved desired activity, or a decreased undesirable activity.Polynucleotides encoding these polypeptide fragments are alsoencompassed by the invention.

In a preferred embodiment, the functional activity displayed by apolypeptide encoded by a polynucleotide fragment of the invention may beone or more biological activities typically associated with thefull-length polypeptide of the invention. Illustrative of thesebiological activities includes the fragments ability to bind to at leastone of the same antibodies which bind to the full-length protein, thefragments ability to interact with at lease one of the same proteinswhich bind to the full-length, the fragments ability to elicit at leastone of the same immune responses as the full-length protein (i.e., tocause the immune system to create antibodies specific to the sameepitope, etc.), the fragments ability to bind to at least one of thesame polynucleotides as the full-length protein, the fragments abilityto bind to a receptor of the full-length protein, the fragments abilityto bind to a ligand of the full-length protein, and the fragmentsability to multimerize with the full-length protein. However, theskilled artisan would appreciate that some fragments may have biologicalactivities which are desirable and directly inapposite to the biologicalactivity of the full-length protein. The functional activity ofpolypeptides of the invention, including fragments, variants,derivatives, and analogs thereof can be determined by numerous methodsavailable to the skilled artisan, some of which are described elsewhereherein.

The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide having anamino acid sequence of SEQ ID NO:2, 24, 54, and/or 56, or an epitope ofthe polypeptide sequence encoded by a polynucleotide sequence containedin ATCC deposit No. Z or encoded by a polynucleotide that hybridizes tothe complement of the sequence of SEQ ID NO:1, 23, 53, and/or 55 orcontained in ATCC deposit No. Z under stringent hybridization conditionsor lower stringency hybridization conditions as defined supra. Thepresent invention further encompasses polynucleotide sequences encodingan epitope of a polypeptide sequence of the invention (such as, forexample, the sequence disclosed in SEQ ID NO:1), polynucleotidesequences of the complementary strand of a polynucleotide sequenceencoding an epitope of the invention, and polynucleotide sequences whichhybridize to the complementary strand under stringent hybridizationconditions or lower stringency hybridization conditions defined supra.

The term “epitopes,” as used herein, refers to portions of a polypeptidehaving antigenic or immunogenic activity in an animal, preferably amammal, and most preferably in a human. In a preferred embodiment, thepresent invention encompasses a polypeptide comprising an epitope, aswell as the polynucleotide encoding this polypeptide. An “immunogenicepitope,” as used herein, is defined as a portion of a protein thatelicits an antibody response in an animal, as determined by any methodknown in the art, for example, by the methods for generating antibodiesdescribed infra. (See, for example, Geysen et al., Proc. Natl. Acad.Sci. USA 81:3998- 4002 (1983)). The term “antigenic epitope,” as usedherein, is defined as a portion of a protein to which an antibody canimmunospecifically bind its antigen as determined by any method wellknown in the art, for example, by the immunoassays described herein.Immunospecific binding excludes non-specific binding but does notnecessarily exclude cross-reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic.

Fragments which function as epitopes may be produced by any conventionalmeans. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135(1985), further described in U.S. Pat. No. 4,631,211).

In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 20, at least 25, atleast 30, at least 40, at least 50, and, most preferably, between about15 to about 30 amino acids. Preferred polypeptides comprisingimmunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acidresidues in length, or longer. Additional non-exclusive preferredantigenic epitopes include the antigenic epitopes disclosed herein, aswell as portions thereof. Antigenic epitopes are useful, for example, toraise antibodies, including monoclonal antibodies, that specificallybind the epitope. Preferred antigenic epitopes include the antigenicepitopes disclosed herein, as well as any combination of two, three,four, five or more of these antigenic epitopes. Antigenic epitopes canbe used as the target molecules in immunoassays. (See, for instance,Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science219:660-666 (1983)).

Similarly, immunogenic epitopes can be used, for example, to induceantibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol.66:2347-2354 (1985). Preferred immunogenic epitopes include theimmunogenic epitopes disclosed herein, as well as any combination oftwo, three, four, five or more of these immunogenic epitopes. Thepolypeptides comprising one or more immunogenic epitopes may bepresented for eliciting an antibody response together with a carrierprotein, such as an albumin, to an animal system (such as rabbit ormouse), or, if the polypeptide is of sufficient length (at least about25 amino acids), the polypeptide may be presented without a carrier.However, immunogenic epitopes comprising as few as 8 to 10 amino acidshave been shown to be sufficient to raise antibodies capable of bindingto, at the very least, linear epitopes in a denatured polypeptide (e.g.,in Western blotting).

Epitope-bearing polypeptides of the present invention may be used toinduce antibodies according to methods well known in the art including,but not limited to, in vivo immunization, in vitro immunization, andphage display methods. See, e.g., Sutcliffe et al., supra; Wilson etal., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). Ifin vivo immunization is used, animals may be immunized with freepeptide; however, anti-peptide antibody titer may be boosted by couplingthe peptide to a macromolecular carrier, such as keyhole limpethemacyanin (KLH) or tetanus toxoid. For instance, peptides containingcysteine residues may be coupled to a carrier using a linker such asmaleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde. Animals such as rabbits, rats and mice are immunizedwith either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μg of peptide or carrier protein and Freund's adjuvant or anyother adjuvant known for stimulating an immune response. Several boosterinjections may be needed, for instance, at intervals of about two weeks,to provide a useful titer of anti-peptide antibody which can bedetected, for example, by ELISA assay using free peptide adsorbed to asolid surface. The titer of anti-peptide antibodies in serum from animmunized animal may be increased by selection of anti-peptideantibodies, for instance, by adsorption to the peptide on a solidsupport and elution of the selected antibodies according to methods wellknown in the art.

As one of skill in the art will appreciate, and as discussed above, thepolypeptides of the present invention comprising an immunogenic orantigenic epitope can be fused to other polypeptide sequences. Forexample, the polypeptides of the present invention may be fused with theconstant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portionsthereof (CH1, CH2, CH3, or any combination thereof and portions thereof)resulting in chimeric polypeptides. Such fusion proteins may facilitatepurification and may increase half-life in vivo. This has been shown forchimeric proteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. See, e.g., EP 394,827;Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of anantigen across the epithelial barrier to the immune system has beendemonstrated for antigens (e.g., insulin) conjugated to an FcRn bindingpartner such as IgG or Fc fragments (see, e.g., PCT Publications WO96/22024 and WO 99/04813). IgG Fusion proteins that have adisulfide-linked dimeric structure due to the IgG portion disulfidebonds have also been found to be more efficient in binding andneutralizing other molecules than monomeric polypeptides or fragmentsthereof alone. See, e.g., Fountoulakis et al., J. Biochem.,270:3958-3964 (1995). Nucleic acids encoding the above epitopes can alsobe recombined with a gene of interest as an epitope tag (e.g., thehemagglutinin (“HA”) tag or flag tag) to aid in detection andpurification of the expressed polypeptide. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972- 897). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag consisting of six histidine residues. The tagserves as a matrix binding domain for the fusion protein. Extracts fromcells infected with the recombinant vaccinia virus are loaded onto Ni2+nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

Additional fusion proteins of the invention may be generated through thetechniques of gene-shuffling, motif-shuffling, exon-shuffling, and/orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling may be employed to modulate the activities of polypeptides ofthe invention, such methods can be used to generate polypeptides withaltered activity, as well as agonists and antagonists of thepolypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238;5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. OpinionBiotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82(1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzoand Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents andpublications are hereby incorporated by reference in its entirety). Inone embodiment, alteration of polynucleotides corresponding to SEQ IDNO:1, 23, 53, and/or 55 and the polypeptides encoded by thesepolynucleotides may be achieved by DNA shuffling. DNA shuffling involvesthe assembly of two or more DNA segments by homologous or site-specificrecombination to generate variation in the polynucleotide sequence. Inanother embodiment, polynucleotides of the invention, or the encodedpolypeptides, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of a polynucleotide encodinga polypeptide of the invention may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

Antibodies

Further polypeptides of the invention relate to antibodies and T-cellantigen receptors (TCR) which immunospecifically bind a polypeptide,polypeptide fragment, or variant of SEQ ID NO:2, 24, 54, and/or 56,and/or an epitope, of the present invention (as determined byimmunoassays well known in the art for assaying specificantibody-antigen binding). Antibodies of the invention include, but arenot limited to, polyclonal, monoclonal, monovalent, bispecific,heteroconjugate, multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab′) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of theinvention), and epitope-binding fragments of any of the above. The term“antibody,” as used herein, refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. The immunoglobulin molecules of the invention can beof any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.Moreover, the term “antibody” (Ab) or “monoclonal antibody” (Mab) ismeant to include intact molecules, as well as, antibody fragments (suchas, for example, Fab and F(ab′)2 fragments) which are capable ofspecifically binding to protein. Fab and F(ab′)2 fragments lack the Fcfragment of intact antibody, clear more rapidly from the circulation ofthe animal or plant, and may have less non-specific tissue binding thanan intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Thus,these fragments are preferred, as well as the products of a FAB or otherimmunoglobulin expression library. Moreover, antibodies of the presentinvention include chimeric, single chain, and humanized antibodies.

Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine (e.g., mouse andrat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.As used herein, “human” antibodies include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., PCT publications WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J.Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Antibodies whichspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homologue of a polypeptide of the presentinvention are included. Antibodies that bind polypeptides with at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 65%, at least 60%, at least 55%, and at least 50% identity(as calculated using methods known in the art and described herein) to apolypeptide of the present invention are also included in the presentinvention. In specific embodiments, antibodies of the present inventioncross-react with murine, rat and/or rabbit homologues of human proteinsand the corresponding epitopes thereof. Antibodies that do not bindpolypeptides with less than 95%, less than 90%, less than 85%, less than80%, less than 75%, less than 70%, less than 65%, less than 60%, lessthan 55%, and less than 50% identity (as calculated using methods knownin the art and described herein) to a polypeptide of the presentinvention are also included in the present invention. In a specificembodiment, the above-described cross-reactivity is with respect to anysingle specific antigenic or immunogenic polypeptide, or combination(s)of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenicpolypeptides disclosed herein. Further included in the present inventionare antibodies which bind polypeptides encoded by polynucleotides whichhybridize to a polynucleotide of the present invention under stringenthybridization conditions (as described herein). Antibodies of thepresent invention may also be described or specified in terms of theirbinding affinity to a polypeptide of the invention. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10-2 M, 10-2 M, 5×10-3 M, 10-3 M, 5×10-4 M, 10-4 M, 5 ×10-5 M, 10-5 M,5×10-6 M, 10-6M, 5×10-7 M, 107 M,5×10-8 M, 10-8 M, 5×10-9 M, 10-9 M,5×10-10 M, 10-10 M, 5 ×10-11 M, 10-11 M, 5×10-12 M, 10-12 M, 5×10-13 M,10-13 M, 5×10-14 M, 10-14 M,5×10-15 M, or 10-15 M.

The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least95%, at least 90%, at least 85 %, at least 80%, at least 75%, at least70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonistsof the polypeptides of the present invention. For example, the presentinvention includes antibodies which disrupt the receptor/ligandinteractions with the polypeptides of the invention either partially orfully. Preferably, antibodies of the present invention bind an antigenicepitope disclosed herein, or a portion thereof. The invention featuresboth receptor-specific antibodies and ligand-specific antibodies. Theinvention also features receptor-specific antibodies which do notprevent ligand binding but prevent receptor activation. Receptoractivation (i.e., signaling) may be determined by techniques describedherein or otherwise known in the art. For example, receptor activationcan be determined by detecting the phosphorylation (e.g., tyrosine orserine/threonine) of the receptor or its substrate byimmunoprecipitation followed by western blot analysis (for example, asdescribed supra). In specific embodiments, antibodies are provided thatinhibit ligand activity or receptor activity by at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least60%, or at least 50% of the activity in absence of the antibody.

The invention also features receptor-specific antibodies which bothprevent ligand binding and receptor activation as well as antibodiesthat recognize the receptor-ligand complex, and, preferably, do notspecifically recognize the unbound receptor or the unbound ligand.Likewise, included in the invention are neutralizing antibodies whichbind the ligand and prevent binding of the ligand to the receptor, aswell as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation, for example, byinducing dimerization of the receptor. The antibodies may be specifiedas agonists, antagonists or inverse agonists for biological activitiescomprising the specific biological activities of the peptides of theinvention disclosed herein. The above antibody agonists can be madeusing methods known in the art. See, e.g., PCT publication WO 96/40281;U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chenet al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214(1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al.,J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol.Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241(1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997);Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996)(which are all incorporated by reference herein in their entireties).

Antibodies of the present invention may be used, for example, but notlimited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionucleotides, or toxins. See,e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat.No. 5,314,995; and EP 396,387.

The antibodies of the invention include derivatives that are modified,i.e., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromgenerating an anti-idiotypic response. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

The antibodies of the present invention may be generated by any suitablemethod known in the art.

The antibodies of the present invention may comprise polyclonalantibodies. Methods of preparing polyclonal antibodies are known to theskilled artisan (Harlow, et al., Antibodies: A Laboratory Manual, (Coldspring Harbor Laboratory Press, 2^(nd) ed. (1988); and CurrentProtocols, Chapter 2; which are hereby incorporated herein by referencein its entirety). In a preferred method, a preparation of the XXXXXprotein is prepared and purified to render it substantially free ofnatural contaminants. Such a preparation is then introduced into ananimal in order to produce polyclonal antisera of greater specificactivity. For example, a polypeptide of the invention can beadministered to various host animals including, but not limited to,rabbits, mice, rats, etc. to induce the production of sera containingpolyclonal antibodies specific for the antigen. The administration ofthe polypeptides of the present invention may entail one or moreinjections of an immunizing agent and, if desired, an adjuvant. Variousadjuvants may be used to increase the immunological response, dependingon the host species, and include but are not limited to, Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants arealso well known in the art. For the purposes of the invention,“immunizing agent” may be defined as a polypeptide of the invention,including fragments, variants, and/or derivatives thereof, in additionto fusions with heterologous polypeptides and other forms of thepolypeptides described herein.

Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections, thoughthey may also be given intramuscularly, and/or through IV). Theimmunizing agent may include polypeptides of the present invention or afusion protein or variants thereof. Depending upon the nature of thepolypeptides (i.e., percent hydrophobicity, percent hydrophilicity,stability, net charge, isoelectric point etc.), it may be useful toconjugate the immunizing agent to a protein known to be immunogenic inthe mammal being immunized. Such conjugation includes either chemicalconjugation by derivitizing active chemical functional groups to boththe polypeptide of the present invention and the immunogenic proteinsuch that a covalent bond is formed, or through fusion-protein basedmethodology, or other methods known to the skilled artisan. Examples ofsuch immunogenic proteins include, but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum. Additionalexamples of adjuvants which may be employed includes the MPL-TDMadjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate).The immunization protocol may be selected by one skilled in the artwithout undue experimentation.

The antibodies of the present invention may comprise monoclonalantibodies. Monoclonal antibodies may be prepared using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975) and U.S. Pat. No. 4,376,110, by Harlow, et al., Antibodies: ALaboratory Manual, (Cold spring Harbor Laboratory Press, 2^(nd) ed.(1988), by Hammerling, et al., Monoclonal Antibodies and T-CellHybridomas (Elsevier, N.Y., pp. 563-681 (1981); K6hler et al., Eur. J.Immunol. 6:511 (1976); Köhler et al., Eur. J. Immunol. 6:292 (1976), orother methods known to the artisan. Other examples of methods which maybe employed for producing monoclonal antibodies includes, but are notlimited to, the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof The hybridoma producingthe mAb of this invention may be cultivated in vitro or in vivo.Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

In a hybridoma method, a mouse, a humanized mouse, a mouse with a humanimmune system, hamster, or other appropriate host animal, is typicallyimmunized with an immunizing agent to elicit lymphocytes that produce orare capable of producing antibodies that will specifically bind to theimmunizing agent. Alternatively, the lymphocytes may be immunized invitro.

The immunizing agent will typically include polypeptides of the presentinvention or a fusion protein thereof. Preferably, the immunizing agentconsists of an XXXXX polypeptide or, more preferably, with a XXXXXpolypeptide-expressing cell. Such cells may be cultured in any suitabletissue culture medium; however, it is preferable to culture cells inEarle's modified Eagle's medium supplemented with 10% fetal bovine serum(inactivated at about 56 degrees C), and supplemented with about 10 g/lof nonessential amino acids, about 1,000 U/ml of penicillin, and about100 ug/ml of streptomycin. Generally, either peripheral bloodlymphocytes (“PBLs”) are used if cells of human origin are desired, orspleen cells or lymph node cells are used if non-human mammalian sourcesare desired. The lymphocytes are then fused with an immortalized cellline using a suitable fusing agent, such as polyethylene glycol, to forma hybridoma cell (Goding, Monoclonal Antibodies: Principles andPractice, Academic Press, (1986), pp. 59-103). Immortalized cell linesare usually transformed mammalian cells, particularly myeloma cells ofrodent, bovine and human origin. Usually, rat or mouse myeloma celllines are employed. The hybridoma cells may be cultured in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, immortalized cells. Forexample, if the parental cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (“HAT medium”), which substances prevent the growth ofHGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. More preferred are the parent myeloma cellline (SP20) as provided by the ATCC. As inferred throughout thespecification, human myeloma and mouse-human heteromyeloma cell linesalso have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against thepolypeptides of the present invention. Preferably, the bindingspecificity of monoclonal antibodies produced by the hybridoma cells isdetermined by immunoprecipitation or by an in vitro binding assay, suchas radioimmunoassay (RIA) or enzyme-linked immunoabsorbant assay(ELISA). Such techniques are known in the art and within the skill ofthe artisan. The binding affmity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis of Munson and Pollart,Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra, and/or according to Wands et al. (Gastroenterology80:225-232 (1981)). Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-sepharose, hydroxyapatite chromatography, gel exclusionchromatography, gel electrophoresis, dialysis, or affinitychromatography.

The skilled artisan would acknowledge that a variety of methods exist inthe art for the production of monoclonal antibodies and thus, theinvention is not limited to their sole production in hydridomas. Forexample, the monoclonal antibodies may be made by recombinant DNAmethods, such as those described in U.S. Pat. No. 4,816,567. In thiscontext, the term “monoclonal antibody” refers to an antibody derivedfrom a single eukaryotic, phage, or prokaryotic clone. The DNA encodingthe monoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies, or such chains from human,humanized, or other sources). The hydridoma cells of the invention serveas a preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transformed into host cells suchas Simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cellsthat do not otherwise produce immunoglobulin protein, to obtain thesynthesis of monoclonal antibodies in the recombinant host cells. TheDNA also may be modified, for example, by substituting the codingsequence for hurman heavy and light chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison etal, supra) or by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. Such a non-immunoglobulin polypeptide can be substitutedfor the constant domains of an antibody of the invention, or can besubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart. Monoclonal antibodies can be prepared using a wide variety oftechniques known in the art including the use of hybridoma, recombinant,and phage display technologies, or a combination thereof. For example,monoclonal antibodies can be produced using hybridoma techniquesincluding those known in the art and taught, for example, in Harlow etal., Antibodies: A Laboratory Manual, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies andT-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said referencesincorporated by reference in their entireties). The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. The term “monoclonal antibody” refers to anantibody that is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art and arediscussed in detail in the Examples described herein. In a non-limitingexample, mice can be immunized with a polypeptide of the invention or acell expressing such peptide. Once an immune response is detected, e.g.,antibodies specific for the antigen are detected in the mouse serum, themouse spleen is harvested and splenocytes isolated. The splenocytes arethen fused by well known techniques to any suitable myeloma cells, forexample cells from cell line SP20 available from the ATCC. Hybridomasare selected and cloned by limited dilution. The hybridoma clones arethen assayed by methods known in the art for cells that secreteantibodies capable of binding a polypeptide of the invention. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by immunizing mice with positive hybridoma clones.

Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CHI domain ofthe heavy chain.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinkman et al., J. Immunol. Methods 182:41-50(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al.,Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994); PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties). Examples of techniques which can be used toproduce single-chain Fvs and antibodies include those described in U.S.Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra etal., Science 240:1038-1040 (1988).

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use chimeric, humanized,or human antibodies. A chimeric antibody is a molecule in whichdifferent portions of the antibody are derived from different animalspecies, such as antibodies having a variable region derived from amurine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; Cabillyet al., Taniguchi et al., EP 171496; Morrison et al., EP 173494;Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne etal., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985);U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which areincorporated herein by reference in their entirety. Humanized antibodiesare antibody molecules from non-human species antibody that binds thedesired antigen having one or more complementarity determining regions(CDRs) from the non-human species and a framework regions from a humanimmunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. (See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature 332:323 (1988), which areincorporated herein by reference in their entireties.) Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332). Generally, a humanized antibody has one or more aminoacid residues introduced into it from a source that is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the methods ofWinter and co-workers (Jones et al., Nature, 321:522-525 (1986);Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534-1536 (1988), by substituting rodent CDRs or CDR sequences forthe corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possible some FR residues aresubstituted from analogous sites in rodent antibodies.

In general, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR regions are thoseof a human immunoglobulin consensus sequence. The humanized antibodyoptimally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin (Jones etal., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-329(1988)1 and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety. The techniques of cole et al., andBoerder et al., are also available for the preparation of humanmonoclonal antibodies (cole et al., Monoclonal Antibodies and CancerTherapy, Alan R. Riss, (1985); and Boerner et al., J. Immunol.,147(1):86-95, (1991)).

Human antibodies can also be produced using transgenic mice which areincapable of expressing fimctional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-fimctional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; and 5,939,598, which are incorporated by referenceherein in their entirety. In addition, companies such as Abgenix, Inc.(Freemont, Calif.), Genpharm (San Jose, Calif.), and Medarex, Inc.(Princeton, N.J.) can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove.

Similarly, human antibodies can be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and creation of an antibody repertoire.This approach is described, for example, in U.S. Pat. Nos. 5,545,807;5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,106, and in thefollowing scientific publications: Marks et al., Biotechnol., 10:779-783(1992); Lonberg et al., Nature 368:856-859 (1994); Fishwild et al.,Nature Biotechnol., 14:845-51 (1996); Neuberger, Nature Biotechnol.,14:826 (1996); Lonberg and Huszer, Intern. Rev. Immunol., 13:65-93(1995).

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

Such anti-idiotypic antibodies capable of binding to the XXXXXpolypeptide can be produced in a two-step procedure. Such a method makesuse of the fact that antibodies are themselves antigens, and therefore,it is possible to obtain an antibody that binds to a second antibody. Inaccordance with this method, protein specific antibodies are used toimmunize an animal, preferably a mouse. The splenocytes of such ananimal are then used to produce hybridoma cells, and the hybridoma cellsare screened to identify clones that produce an antibody whose abilityto bind to the protein-specific antibody can be blocked by thepolypeptide. Such antibodies comprise anti-idiotypic antibodies to theprotein-specific antibody and can be used to immunize an animal toinduce formation of fuirther protein-specific antibodies.

The antibodies of the present invention may be bispecific antibodies.Bispecific antibodies are monoclonal, Preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present invention, one of the binding specificities maybe directed towards a polypeptide of the present invention, the othermay be for any other antigen, and preferably for a cell-surface protein,receptor, receptor subunit, tissue-specific antigen, virally derivedprotein, virally encoded envelope protein, bacterially derived protein,or bacterial surface protein, etc.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatographysteps. Similar procedures are disclosed in WO 93/08829, published 13 May1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transformed into a suitable host organism. Forfurther details of generating bispecific antibodies see, for exampleSuresh et al., Meth. In Enzym., 121:210 (1986).

Heteroconjugate antibodies are also contemplated by the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4, 676,980), and for the treatment of HIV infection (WO 91/00360; WO 92/20373;and EP03089). It is contemplated that the antibodies may be prepared invitro using known methods in synthetic protein chemistry, includingthose involving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioester bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides comprising a nucleotidesequence encoding an antibody of the invention and fragments thereof.The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions, e.g., as definedsupra, to polynucleotides that encode an antibody, preferably, thatspecifically binds to a polypeptide of the invention, preferably, anantibody that binds to a polypeptide having the amino acid sequence ofSEQ ID NO:2, 24, 54, and/or 56.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, or nucleic acid, preferably poly A+ RNA, isolated from, any tissueor cells expressing the antibody, such as hybridoma cells selected toexpress an antibody of the invention) by PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence orby cloning using an oligonucleotide probe specific for the particulargene sequence to identify, e.g., a cDNA clone from a cDNA library thatencodes the antibody. Amplified nucleic acids generated by PCR may thenbe cloned into replicable cloning vectors using any method well known inthe art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY, which are both incorporated by reference herein in theirentireties ), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell know in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody, asdescribed supra. The framework regions may be naturally occurring orconsensus framework regions, and preferably human framework regions(see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for alisting of human framework regions). Preferably, the polynucleotidegenerated by the combination of the framework regions and CDRs encodesan antibody that specifically binds a polypeptide of the invention.Preferably, as discussed supra, one or more amino acid substitutions maybe made within the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibody to its antigen.Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423- 42 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Wardet al., Nature 334:544-54 (1989)) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,Science 242:1038- 1041 (1988)).

More preferably, a clone encoding an antibody of the present inventionmay be obtained according to the method described in the Example sectionherein.

Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment,derivative or analog thereof, (e.g., a heavy or light chain of anantibody of the invention or a single chain antibody of the invention),requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, or a single chainantibody of the invention, operably linked to a heterologous promoter.In preferred embodiments for the expression of double-chainedantibodies, vectors encoding both the heavy and light chains may beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts. (e.g., see Logan &Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., Methodsin Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, W138, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can beemployed in tk-, hgprt- or aprt- cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods commonly known in the art of recombinant DNA technology may beroutinely applied to select the desired recombinant clone, and suchmethods are described, for example, in Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), CurrentProtocols in Human Genetics, John Wiley & Sons, NY (1994);Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol.3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressing,both heavy and light chain polypeptides. In such situations, the lightchain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc.Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavyand light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention to generate fusion proteins. Thefusion does not necessarily need to be direct, but may occur throughlinker sequences. The antibodies may be specific for antigens other thanpolypeptides (or portion thereof, preferably at least 10, 20, 30, 40,50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the presentinvention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol.146:2446-2452(1991), which are incorporated by reference in theirentireties.

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the constant region, hinge region,CH1 domain, CH2 domain, and CH3 domain or any combination of wholedomains or portions thereof. The polypeptides may also be fused orconjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. lrmunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337-11341(1992) (said references incorporated by reference in theirentireties).

As discussed, supra, the polypeptides corresponding to a polypeptide,polypeptide fragment, or a variant of SEQ ID NO:2, 24, 54, and/or 56 maybe fused or conjugated to the above antibody portions to increase the invivo half life of the polypeptides or for use in immunoassays usingmethods known in the art. Further, the polypeptides corresponding to SEQID NO:2, 24, 54, and/or 56 may be fused or conjugated to the aboveantibody portions to facilitate purification. One reported exampledescribes chimeric proteins consisting of the first two domains of thehuman CD4-polypeptide and various domains of the constant regions of theheavy or light chains of mammalian immunoglobulins. (EP 394,827;Traunecker et al., Nature 331:84-86 (1988). The polypeptides of thepresent invention fused or conjugated to an antibody havingdisulfide-linked dimeric structures (due to the IgG) may also be moreefficient in binding and neutralizing other molecules, than themonomeric secreted protein or protein fragment alone. (Fountoulakis etal., J. Biochem. 270:3958-3964 (1995)). In many cases, the Fc part in afusion protein is beneficial in therapy and diagnosis, and thus canresult in, for example, improved pharmacokinetic properties. (EP A232,262). Alternatively, deleting the Fc part after the fusion proteinhas been expressed, detected, and purified, would be desired. Forexample, the Fc portion may hinder therapy and diagnosis if the fusionprotein is used as an antigen for immunizations. In drug discovery, forexample, human proteins, such as hIL-5, have been fused with Fc portionsfor the purpose of high-throughput screening assays to identifyantagonists of hIL-5. (See, Bennett et al., J. Molecular Recognition8:52-58 (1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide to facilitatepurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude 125I, 131I, 111In or 99Tc.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologues thereof. Therapeutic agents include, but are not limitedto, antimetabolites (e.g., methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylatingagents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin(formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)),and anti-mitotic agents (e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, α-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No.WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

The present invention also encompasses the creation of syntheticantibodies directed against the polypeptides of the present invention.One example of synthetic antibodies is described in Radrizzani, M., etal., Medicina, (Aires), 59(6):753-8, (1999)). Recently, a new class ofsynthetic antibodies has been described and are referred to asmolecularly imprinted polymers (MIPs) (Semorex, Inc.). Antibodies,peptides, and enzymes are often used as molecular recognition elementsin chemical and biological sensors. However, their lack of stability andsignal transduction mechanisms limits their use as sensing devices.Molecularly imprinted polymers (MIPs) are capable of mimicking thefunction of biological receptors but with less stability constraints.Such polymers provide high sensitivity and selectivity while maintainingexcellent thermal and mechanical stability. MIPs have the ability tobind to small molecules and to target molecules such as organics andproteins' with equal or greater potency than that of natural antibodies.These “super” MIPs have higher affinities for their target and thusrequire lower concentrations for efficacious binding.

During synthesis, the MIPs are imprinted so as to have complementarysize, shape, charge and functional groups of the selected target byusing the target molecule itself (such as a polypeptide, antibody,etc.), or a substance having a very similar structure, as its “print” or“template.” MIPs can be derivatized with the same reagents afforded toantibodies. For example, fluorescent ‘super’ MIPs can be coated ontobeads or wells for use in highly sensitive separations or assays, or foruse in high throughput screening of proteins.

Moreover, MIPs based upon the structure of the polypeptide(s) of thepresent invention may be useful in screening for compounds that bind tothe polypeptide(s) of the invention. Such a MIP would serve the role ofa synthetic “receptor” by minimicking the native architecture of thepolypeptide. In fact, the ability of a MIP to serve the role of asynthetic receptor has already been demonstrated for the estrogenreceptor (Ye, L., Yu, Y., Mosbach, K, Analyst., 126(6):760-5, (2001);Dickert, F, L., Hayden, O., Halilias, K, P, Analyst., 126(6):766-71,(2001)). A synthetic receptor may either be mimicked in its entirety(e.g., as the entire protein), or mimicked as a series of short peptidescorresponding to the protein (Rachkov, A., Minoura, N, Biochim, Biophys,Acta., 1544(1-2):255-66, (2001)). Such a synthetic receptor MIPs may beemployed in any one or more of the screening methods described elsewhereherein.

MIPs have also been shown to be useful in “sensing” the presence of itsmimicked molecule (Cheng, Z., Wang, E., Yang, X, Biosens, Bioelectron.,16(3):179-85, (2001); Jenkins, A, L., Yin, R., Jensen, J. L, Analyst.,126(6):798-802, (2001); Jenkins, A, L., Yin, R., Jensen, J. L, Analyst.,126(6):798-802, (2001)). For example, a MIP designed using a polypeptideof the present invention may be used in assays designed to identify, andpotentially quantitate, the level of said polypeptide in a sample. Sucha MIP may be used as a substitute for any component described in theassays, or kits, provided herein (e.g., ELISA, etc.).

A number of methods may be employed to create MIPs to a specificreceptor, ligand, polypeptide, peptide, organic molecule. Severalpreferred methods are described by Esteban et al in J. Anal, Chem.,370(7):795-802, (2001), which is hereby incorporated herein by referencein its entirety in addition to any references cited therein. Additionalmethods are known in the art and are encompassed by the presentinvention, such as for example, Hart, B, R., Shea, K, J. J. Am. Chem,Soc., 123(9):2072-3, (2001); and Quaglia, M., Chenon, K., Hall, A, J.,De, Lorenzi, E., Sellergren, B, J. Am. Chem, Soc., 123(10):2146-54,(2001); which are hereby incorporated by reference in their entiretyherein.

Uses for Antibodies Directed Against Polypeptides of the Invention

The antibodies of the present invention have various utilities. Forexample, such antibodies may be used in diagnostic assays to detect thepresence or quantification of the polypeptides of the invention in asample. Such a diagnostic assay may be comprised of at least two steps.The first, subjecting a sample with the antibody, wherein the sample isa tissue (e.g., human, animal, etc.), biological fluid (e.g., blood,urine, sputum, semen, amniotic fluid, saliva, etc.), biological extract(e.g., tissue or cellular homogenate, etc.), a protein microchip (e.g.,See Arenkov P, et al., Anal Biochem., 278(2):123-131 (2000)), or achromatography column, etc. And a second step involving thequantification of antibody bound to the substrate. Alternatively, themethod may additionally involve a first step of attaching the antibody,either covalently, electrostatically, or reversibly, to a solid support,and a second step of subjecting the bound antibody to the sample, asdefmed above and elsewhere herein.

Various diagnostic assay techniques are known in the art, such ascompetitive binding assays, direct or indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogenous phases (Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc., (1987), ppl47-158). The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as 2H, 14C, 32P, or 125I, a florescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase, green fluorescent protein, or horseradishperoxidase. Any method known in the art for conjugating the antibody tothe detectable moiety may be employed, including those methods describedby Hunter et al., Nature, 144:945 (1962); Dafvid et al., Biochem.,13:1014 (1974); Pain et al., J. Immunol. Metho., 40:219(1981); andNygren, J. Histochem. And Cytochem., 30:407 (1982).

Antibodies directed against the polypeptides of the present inventionare useful for the affinity purification of such polypeptides fromrecombinant cell culture or natural sources. In this process, theantibodies against a particular polypeptide are immobilized on asuitable support, such as a Sephadex resin or filter paper, usingmethods well known in the art. The immobilized antibody then iscontacted with a sample containing the polypeptides to be purified, andthereafter the support is washed with a suitable solvent that willremove substantially all the material in the sample except for thedesired polypeptides, which are bound to the immobilized antibody.Finally, the support is washed with another suitable solvent that willrelease the desired polypeptide from the antibody.

Immunophenotyping

The antibodies of the invention may be utilized for immunophenotyping ofcell lines and biological samples. The translation product of the geneof the present invention may be usefuil as a cell specific marker, ormore specifically as a cellular marker that is differentially expressedat various stages of differentiation and/or maturation of particularcell types. Monoclonal antibodies directed against a specific epitope,or combination of epitopes, will allow for the screening of cellularpopulations expressing the marker. Various techniques can be utilizedusing monoclonal antibodies to screen for cellular populationsexpressing the marker(s), and include magnetic separation usingantibody-oated magnetic beads, “panning” with antibody attached to asolid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No.5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

These techniques allow for the screening of particular populations ofcells, such as might be found with hematological malignancies (i.e.minimal residual disease (MRD) in acute leukemic patients) and“non-self” cells in transplantations to prevent Graft-versus-HostDisease (GVHD). Alternatively, these techniques allow for the screeningof hematopoietic stem and progenitor cells capable of undergoingproliferation and/or differentiation, as might be found in humanumbilical cord blood.

Assays For Antibody Binding

The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-ompetitive assaysystems using techniques such as western blots, radioimmunoassays, ELISA(enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP40 or Triton X-100, 1%sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate atpH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 14 hours) at 4° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., 32P or 125I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., 3H or 125I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest conjugated to a labeled compound (e.g., 3H or 125I)in the presence of increasing amounts of an unlabeled second antibody.

Therapeutic Uses of Antibodies

The present invention is further directed to antibody-based therapieswhich involve administering antibodies of the invention to an animal,preferably a mammal, and most preferably a human, patient for treatingone or more of the disclosed diseases, disorders, or conditions.Therapeutic compounds of the invention include, but are not limited to,antibodies of the invention (including fragments, analogs andderivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).The antibodies of the invention can be used to treat, inhibit or preventdiseases, disorders or conditions associated with aberrant expressionand/or activity of a polypeptide of the invention, including, but notlimited to, any one or more of the diseases, disorders, or conditionsdescribed herein. The treatment and/or prevention of diseases,disorders, or conditions associated with aberrant expression and/oractivity of a polypeptide of the invention includes, but is not limitedto, alleviating symptoms associated with those diseases, disorders orconditions. Antibodies of the invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

It is preferred to use high affmity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affmity for polynucleotides or polypeptides of theinvention, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10-2 M,10-2 M, 5×10-3 M, 10-3 M, 5×10-4 M, 10-4 M, 5×10-5 M, 10-5 M, 5×10-6 M,10-6 M, 5×10-7 M, 10-7 M, 5×10-8 M, 10-8 M, 5×10-9 M, 10-9 M, 5×10-10 M,10-10 M, 5×10-11 M, 10-11 M, 5×10-12 M, 10-12 M, 5×10-13 M, 10-13 M,5×10-14 M, 10-14 M, 5×10-15 M, and 10-15 M.

Antibodies directed against polypeptides of the present invention areuseful for inhibiting allergic reactions in animals. For example, byadministering a therapeutically acceptable dose of an antibody, orantibodies, of the present invention, or a cocktail of the presentantibodies, or in combination with other antibodies of varying sources,the animal may not elicit an allergic response to antigens.

Likewise, one could envision cloning the gene encoding an antibodydirected against a polypeptide of the present invention, saidpolypeptide having the potential to elicit an allergic and/or immuneresponse in an organism, and transforming the organism with saidantibody gene such that it is expressed (e.g., constitutively,inducibly, etc.) in the organism. Thus, the organism would effectivelybecome resistant to an allergic response resulting from the ingestion orpresence of such an immune/allergic reactive polypeptide. Moreover, sucha use of the antibodies of the present invention may have particularutility in preventing and/or ameliorating autoimmune diseases and/ordisorders, as such conditions are typically a result of antibodies beingdirected against endogenous proteins. For example, in the instance wherethe polypeptide of the present invention is responsible for modulatingthe immune response to auto-antigens, transforming the organism and/orindividual with a construct comprising any of the promoters disclosedherein or otherwise known in the art, in addition, to a polynucleotideencoding the antibody directed against the polypeptide of the presentinvention could effective inhibit the organisms immune system fromeliciting an immune response to the auto-antigen(s). Detaileddescriptions of therapeutic and/or gene therapy applications of thepresent invention are provided elsewhere herein.

Alternatively, antibodies of the present invention could be produced ina plant (e.g., cloning the gene of the antibody directed against apolypeptide of the present invention, and transforming a plant with asuitable vector comprising said gene for constitutive expression of theantibody within the plant), and the plant subsequently ingested by ananimal, thereby conferring temporary immunity to the animal for thespecific antigen the antibody is directed towards (See, for example,U.S. Pat. Nos. 5,914,123 and 6,034,298).

In another embodiment, antibodies of the present invention, preferablypolyclonal antibodies, more preferably monoclonal antibodies, and mostpreferably single-chain antibodies, can be used as a means of inhibitinggene expression of a particular gene, or genes, in a human, mammal,and/or other organism. See, for example, International PublicationNumber WO 00/05391, published Feb. 3, 2000, to Dow Agrosciences LLC. Theapplication of such methods for the antibodies of the present inventionare known in the art, and are more particularly described elsewhereherein.

In yet another embodiment, antibodies of the present invention may beuseful for multimerizing the polypeptides of the present invention. Forexample, certain proteins may confer enhanced biological activity whenpresent in a multimeric state (i.e., such enhanced activity may be dueto the increased effective concentration of such proteins whereby moreprotein is available in a localized location).

Antibody-Based Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingantibodies or functional derivatives thereof, are administered to treat,inhibit or prevent a disease or disorder associated with aberrantexpression and/or activity of a polypeptide of the invention, by way ofgene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95(1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993);Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, the compound comprises nucleic acid sequencesencoding an antibody, said nucleic acid sequences being part ofexpression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody encodingnucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); Zijlstra et al., Nature 342:435438 (1989). Inspecific embodiments, the expressed antibody molecule is a single chainantibody; alternatively, the nucleic acid sequences include sequencesencoding both the heavy and light chains, or fragments thereof, of theantibody.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid- carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by adrninistering themin linkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635;WO92/20316; WO93114188, WO 93/20221). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature342:435-438 (1989)).

In a specific embodiment, viral vectors that contains nucleic acidsequences encoding an antibody of the invention are used. For example, aretroviral vector can be used (see Miller et al., Meth. Enzymol.217:581-599 (1993)). These retroviral vectors contain the componentsnecessary for the correct packaging of the viral genome and integrationinto the host cell DNA. The nucleic acid sequences encoding the antibodyto be used in gene therapy are cloned into one or more vectors, whichfacilitates delivery of the gene into a patient. More detail aboutretroviral vectors can be found in Boesen et al., Biotherapy 6:291-302(1994), which describes the use of a retroviral vector to deliver themdrl gene to hematopoietic stem cells in order to make the stem cellsmore resistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest.93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons andGunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson,Curr. Opin. in Genetics and Devel. 3:110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In apreferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993);U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asTlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody are introduced into thecells such that they are expressible by the cells or their progeny, andthe recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598; Stemple andAnderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229(1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription. Demonstration of Therapeutic or Prophylactic Activity Thecompounds or pharmaceutical compositions of the invention are preferablytested in vitro, and then in vivo for the desired therapeutic orprophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

Therapeutic/Prophylactic Administration and Compositions

The invention provides methods of treatment, inhibition and prophylaxisby administration to a subject of an effective amount of a compound orpharmaceutical composition of the invention, preferably an antibody ofthe invention. In a preferred aspect, the compound is substantiallypurified (e.g., substantially free from substances that limit its effector produce undesired side-effects). The subject is preferably an animal,including but not limited to animals such as cows, pigs, horses,chickens, cats, dogs, etc., and is preferably a mammal, and mostpreferably human.

Formulations and methods of administration that can be employed when thecompound comprises a nucleic acid or an immunoglobulin are describedabove; additional appropriate formulations and routes of administrationcan be selected from among those described herein below.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

In another embodiment, the compound or composition can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.)

In yet another embodiment, the compound or composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, N.Y.(1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61(1983); see also Levy et al., Science 228:190 (1985); During et al.,Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)).In yet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., the brain, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a compound,and a pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with - cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the compound of the invention which will be effective inthe treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of a polypeptide ofthe invention can be determined by standard clinical techniques. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

Diagnosis and Imaging with Antibodies

Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases, disorders,and/or conditions associated with the aberrant expression and/oractivity of a polypeptide of the invention. The invention provides forthe detection of aberrant expression of a polypeptide of interest,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosing a disorder,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of a particular disorder.With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Antibodies of the invention can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell . Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (1251, 1211), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a diseaseor disorder associated with aberrant expression of a polypeptide ofinterest in an animal, preferably a mammal and most preferably a human.In one embodiment, diagnosis comprises: a) administering (for example,parenterally, subcutaneously, or intraperitoneally) to a subject aneffective amount of a labeled molecule which specifically binds to thepolypeptide of interest; b) waiting for a time interval following theadministering for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject where the polypeptide is expressed(and for unbound labeled molecule to be cleared to background level); c)determining background level; and d) detecting the labeled molecule inthe subject, such that detection of labeled molecule above thebackground level indicates that the subject has a particular disease ordisorder associated with aberrant expression of the polypeptide ofinterest. Background level can be determined by various methodsincluding, comparing the amount of labeled molecule detected to astandard value previously determined for a particular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of 99mTc. The labeled antibody orantibody fragment will then preferentially accumulate at the location ofcells which contain the specific protein. In vivo tumor imaging isdescribed in S. W. Burchiel et al., “lrmunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Kits

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

In another specific embodiment of the present invention, the kit is adiagnostic kit for use in screening serum containing antibodies specificagainst proliferative and/or cancerous polynucleotides and polypeptides.Such a kit may include a control antibody that does not react with thepolypeptide of interest. Such a kit may include a substantially isolatedpolypeptide antigen comprising an epitope which is specificallyimmunoreactive with at least one anti-polypeptide antigen antibody.Further, such a kit includes means for detecting the binding of saidantibody to the antigen (e.g., the antibody may be conjugated to afluorescent compound such as fluorescein or rhodamine which can bedetected by flow cytometry). In specific embodiments, the kit mayinclude a recombinantly produced or chemically synthesized polypeptideantigen. The polypeptide antigen of the kit may also be attached to asolid support.

In a more specific embodiment the detecting means of the above-describedkit includes a solid support to which said polypeptide antigen isattached. Such a kit may also include a non-attached reporter-labeledanti-human antibody. In this embodiment, binding of the antibody to thepolypeptide antigen can be detected by binding of the saidreporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with polypeptide or polynucleotide antigens,and means for detecting the binding of the polynucleotide or polypeptideantigen to the antibody. In one embodiment, the antibody is attached toa solid support. In a specific embodiment, the antibody may be amonoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention. After binding with specific antigen antibody tothe reagent and removing unbound serum components by washing, thereagent is reacted with reporter-labeled anti-human antibody to bindreporter to the reagent in proportion to the amount of boundanti-antigen antibody on the solid support. The reagent is again washedto remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes a support withsurface- bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

Fusion Proteins

Any polypeptide of the present invention can be used to generate fusionproteins. For example, the polypeptide of the present invention, whenfused to a second protein, can be used as an antigenic tag. Antibodiesraised against the polypeptide of the present invention can be used toindirectly detect the second protein by binding to the polypeptide.Moreover, because certain proteins target cellular locations based ontrafficking signals, the polypeptides of the present invention can beused as targeting molecules once fused to other proteins.

Examples of domains that can be fused to polypeptides of the presentinvention include not only heterologous signal sequences, but also otherheterologous functional regions. The fusion does not necessarily need tobe direct, but may occur through linker sequences.

Moreover, fusion proteins may also be engineered to improvecharacteristics of the polypeptide of the present invention. Forinstance, a region of additional amino acids, particularly charged aminoacids, may be added to the N-terminus of the polypeptide to improvestability and persistence during purification from the host cell orsubsequent handling and storage. Peptide moieties may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. Similarly, peptidecleavage sites can be introduced in-between such peptide moieties, whichcould additionally be subjected to protease activity to remove saidpeptide(s) from the protein of the present invention. The addition ofpeptide moieties, including peptide cleavage sites, to facilitatehandling of polypeptides are familiar and routine techniques in the art.

Moreover, polypeptides of the present invention, including fragments,and specifically epitopes, can be combined with parts of the constantdomain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1,CH2, CH3, and any combination thereof, including both entire domains andportions thereof), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. One reported example describes chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86(1988).) Fusion proteins having disulfide-linked dimeric structures (dueto the IgG) can also be more efficient in binding and neutralizing othermolecules, than the monomeric secreted protein or protein fragmentalone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).)Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) disclosesfusion proteins comprising various portions of the constant region ofimmunoglobulin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995).)

Moreover, the polypeptides of the present invention can be fused tomarker sequences (also referred to as “tags”). Due to the availabilityof antibodies specific to such “tags”, purification of the fusedpolypeptide of the invention, and/or its identification is significantlyfacilitated since antibodies specific to the polypeptides of theinvention are not required. Such purification may be in the form of anaffinity purification whereby an anti-tag antibody or another type ofaffinity matrix (e.g., anti-tag antibody attached to the matrix of aflow-thru column) that binds to the epitope tag is present. In preferredembodiments, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 EtonAvenue, Chatsworth, Calif., 91311), among others, many of which arecommercially available. As described in Gentz et al., Proc. Natl. Acad.Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides forconvenient purification of the fusion protein. Another peptide taguseful for purification, the “HA” tag, corresponds to an epitope derivedfrom the influenza hemagglutinin protein. (Wilson et al., Cell 37:767(1984)).

The skilled artisan would acknowledge the existence of other “tags”which could be readily substituted for the tags referred to supra forpurification and/or identification of polypeptides of the presentinvention (Jones C., et al., J Chromatogr A. 707(1):3-22 (1995)). Forexample, the c-myc tag and the 8F9, 3C7, 6E10, G4m B7 and 9E10antibodies thereto (Evan et al., Molecular and Cellular Biology5:3610-3616 (1985)); the Herpes Simplex virus glycoprotein D (gD) tagand its antibody (Paborsky et al., Protein Engineering, 3(6):547-553(1990), the Flag-peptide—i.e., the octapeptide sequence DYKDDDDK (SEQ IDNO:47), (Hopp et al., Biotech. 6:1204-1210 (1988); the KT3 epitopepeptide (Martin et al., Science, 255:192-194 (1992)); a-tubulin epitopepeptide (Skinner et al., J. Biol. Chem., 266:15136-15166, (1991)); theT7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc. Natl. Sci.USA, 87:6363-6397 (1990)), the FITC epitope (Zymed, Inc.), the GFPepitope (Zymed, Inc.), and the Rhodamine epitope (Zymed, Inc.).

The present invention also encompasses the attachment of up to ninecodons encoding a repeating series of up to nine arginine amino acids tothe coding region of a polynucleotide of the present invention. Theinvention also encompasses chemically derivitizing a polypeptide of thepresent invention with a repeating series of up to nine arginine aminoacids. Such a tag, when attached to a polypeptide, has recently beenshown to serve as a universal pass, allowing compounds access to theinterior of cells without additional derivitization or manipulation(Wender, P., et al., unpublished data).

Protein fusions involving polypeptides of the present invention,including fragrnents and/or variants thereof, can be used for thefollowing, non-limiting examples, subcellular localization of proteins,determination of protein-protein interactions via immunoprecipitation,purification of proteins via affinity chromatography, functional and/orstructural characterization of protein. The present invention alsoencompasses the application of hapten specific antibodies for any of theuses referenced above for epitope fusion proteins. For example, thepolypeptides of the present invention could be chemically derivatized toattach hapten molecules (e.g., DNP, (Zymed, Inc.)). Due to theavailability of monoclonal antibodies specific to such haptens, theprotein could be readily purified using immunoprecipation, for example.

Polypeptides of the present invention, including fragments and/orvariants thereof, in addition to, antibodies directed against suchpolypeptides, fragments, and/or variants, may be fused to any of anumber of known, and yet to be determined, toxins, such as ricin,saporin (Mashiba H. et al., Ann. N. Y. Acad. Sci. 1999;886:233-5), or HCtoxin (Tonukari N J, et al., Plant Cell. 2000 February;12(2):237-248),for example. Such fusions could be used to deliver the toxins to desiredtissues for which a ligand or a protein capable of binding to thepolypeptides of the invention exists.

The invention encompasses the fusion of antibodies directed againstpolypeptides of the present invention, including variants and fragmentsthereof, to said toxins for delivering the toxin to specific locationsin a cell, to specific tissues, and/or to specific species. Suchbifunctional antibodies are known in the art, though a review describingadditional advantageous fusions, including citations for methods ofproduction, can be found in P. J. Hudson, Curr. Opp. In. Imm.11:548-557, (1999); this publication, in addition to the referencescited therein, are hereby incorporated by reference in their entiretyherein. In this context, the term “toxin” may be expanded to include anyheterologous protein, a small molecule, radionucleotides, cytotoxicdrugs, liposomes, adhesion molecules, glycoproteins, ligands, cell ortissue-specific ligands, enzymes, of bioactive agents, biologicalresponse modifiers, anti-fingal agents, hormones, steroids, vitamins,peptides, peptide analogs, anti-allergenic agents, anti-tubercularagents, anti-viral agents, antibiotics, anti-protozoan agents, chelates,radioactive particles, radioactive ions, X-ray contrast agents,monoclonal antibodies, polyclonal antibodies and genetic material. Inview of the present disclosure, one skilled in the art could determinewhether any particular “toxin” could be used in the compounds of thepresent invention. Examples of suitable “toxins” listed above areexemplary only and are not intended to limit the “toxins” that may beused in the present invention.

Thus, any of these above fusions can be engineered using thepolynucleotides or the polypeptides of the present invention.

Vectors, Host Cells, and Protein Production

The present invention also relates to vectors containing thepolynucleotide of the present invention, host cells, and the productionof polypeptides by recombinant techniques. The vector may be, forexample, a phage, plasmid, viral, or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The polynucleotide insert should be operatively linked to an appropriatepromoter, such as the phage lambda PL promoter, the E. coli lac, trp,phoA and tac promoters, the SV40 early and late promoters and promotersof retroviral LTRs, to name a few. Other suitable promoters will beknown to the skilled artisan. The expression constructs will furthercontain sites for transcription initiation, termination, and, in thetranscribed region, a ribosome binding site for translation. The codingportion of the transcripts expressed by the constructs will preferablyinclude a translation initiating codon at the beginning and atermination codon (UAA, UGA or UAG) appropriately positioned at the endof the polypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase,G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCCAccession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowesmelanoma cells; and plant cells. Appropriate culture mediums andconditions for the above-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from QIAGEN, Inc; pBluescript vectors, Phagescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from StratageneCloning Systems, Inc; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia Biotech, Inc. Among preferred eukaryoticvectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available fromStratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.Preferred expression vectors for use in yeast systems include, but arenot limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ,pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, andPAO815 (all available from Invitrogen, Carlsbad, Calif.). Other suitablevectors will be readily apparent to the skilled artisan.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection, or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986). It is specifically contemplated that the polypeptides ofthe present invention may in fact be expressed by a host cell lacking arecombinant vector.

A polypeptide of this invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.

Polypeptides of the present invention, and preferably the secreted form,can also be recovered from: products purified from natural sources,including bodily fluids, tissues and cells, whether directly isolated orcultured; products of chemical synthetic procedures; and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect,and mammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes. Thus, it is wellknown in the art that the N-terminal methionine encoded by thetranslation initiation codon generally is removed with high efficiencyfrom any protein after translation in all eukaryotic cells. While theN-terminal methionine on most proteins also is efficiently removed inmost prokaryotes, for some proteins, this prokaryotic removal process isinefficient, depending on the nature of the amino acid to which theN-terminal methionine is covalently linked.

In one embodiment, the yeast Pichia pastoris is used to express thepolypeptide of the present invention in a eukaryotic system. Pichiapastoris is a methylotrophic yeast which can metabolize methanol as itssole carbon source. A main step in the methanol metabolization pathwayis the oxidation of methanol to formaldehyde using O2. This reaction iscatalyzed by the enzyme alcohol oxidase. In order to metabolize methanolas its sole carbon source, Pichia pastoris must generate high levels ofalcohol oxidase due, in part, to the relatively low affinity of alcoholoxidase for O2. Consequently, in a growth medium depending on methanolas a main carbon source, the promoter region of one of the two alcoholoxidase genes (AOX1) is highly active. In the presence of methanol,alcohol oxidase produced from the AOX1 gene comprises up toapproximately 30% of the total soluble protein in Pichia pastoris. See,Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, etal., Yeast 5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res.15:3859-76 (1987). Thus, a heterologous coding sequence, such as, forexample, a polynucleotide of the present invention, under thetranscriptional regulation of all or part of the AOX1 regulatorysequence is expressed at exceptionally high levels in Pichia yeast grownin the presence of methanol.

In one example, the plasmid vector pPIC9K is used to express DNAencoding a polypeptide of the invention, as set forth herein, in aPichea yeast system essentially as described in “Pichia Protocols:Methods in Molecular Biology,” D.R. Higgins and J. Cregg, eds. TheHumana Press, Totowa, N.J., 1998. This expression vector allowsexpression and secretion of a protein of the invention by virtue of thestrong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase(PHO) secretory signal peptide (i.e., leader) located upstream of amultiple cloning site.

Many other yeast vectors could be used in place of pPIC9K, such as,pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9,pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815, as one skilled in theart would readily appreciate, as long as the proposed expressionconstruct provides appropriately located signals for transcription,translation, secretion (if desired), and the like, including an in-frameAUG, as required.

In another embodiment, high-level expression of a heterologous codingsequence, such as, for example, a polynucleotide of the presentinvention, may be achieved by cloning the heterologous polynucleotide ofthe invention into an expression vector such as, for example, pGAPZ orpGAPZalpha, and growing the yeast culture in the absence of methanol.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., coding sequence), and/or to include geneticmaterial (e.g., heterologous polynucleotide sequences) that is operablyassociated with the polynucleotides of the invention, and whichactivates, alters, and/or amplifies endogenous polynucleotides. Forexample, techniques known in the art may be used to operably associateheterologous control regions (e.g., promoter and/or enhancer) andendogenous polynucleotide sequences via homologous recombination,resulting in the formation of a new transcription unit (see, e.g., U.S.Pat. No. 5,641,670, issued Jun. 24, 1997; U.S. Pat. No. 5,733,761,issued Mar. 31, 1998; International Publication No. WO 96/29411,published Sep. 26, 1996; International Publication No. WO 94/12650,published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), thedisclosures of each of which are incorporated by reference in theirentireties).

In addition, polypeptides of the invention can be chemically synthesizedusing techniques known in the art (e.g., see Creighton, 1983, Proteins:Structures and Molecular Principles, W.H. Freeman & Co., N.Y., andHunkapiller et al., Nature, 310:105-111 (1984)). For example, apolypeptide corresponding to a fragment of a polypeptide sequence of theinvention can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into thepolypeptide sequence. Non-classical amino acids include, but are notlimited to, to the D-isomers of the common amino acids,2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acidssuch as b-methyl amino acids, Ca-methyl amino acids, Na-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

The invention encompasses polypeptides which are differentially modifiedduring or after translation, e.g., by glycosylation, acetylation,phosphorylation, amidation, derivatization by known protectingiblockinggroups, proteolytic cleavage, linkage to an antibody molecule or othercellular ligand, etc. Any of numerous chemical modifications may becarried out by known techniques, including but not limited, to specificchemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH4; acetylation, formylation, oxidation, reduction;metabolic synthesis in the presence of tunicamycin; etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of prokaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein, the addition ofepitope tagged peptide fragments (e.g., FLAG, HA, GST, thioredoxin,maltose binding protein, etc.), attachment of affinity tags such asbiotin and/or streptavidin, the covalent attachment of chemical moietiesto the amino acid backbone, N- or C-terminal processing of thepolypeptides ends (e.g., proteolytic processing), deletion of theN-terminal methionine residue, etc.

Also provided by the invention are chemically modified derivatives ofthe polypeptides of the invention which may provide additionaladvantages such as increased solubility, stability and circulating timeof the polypeptide, or decreased immunogenicity (see U.S. Pat. No.4,179,337). The chemical moieties for derivitization may be selectedfrom water soluble polymers such as polyethylene glycol, ethyleneglycolipropylene glycol copolymers, carboxymethylcellulose, dextran,polyvinyl alcohol and the like. The polypeptides may be modified atrandom positions within the molecule, or at predetermined positionswithin the molecule and may include one, two, three or more attachedchemical moieties.

The invention further encompasses chemical derivitization of thepolypeptides of the present invention, preferably where the chemical isa hydrophilic polymer residue. Exemplary hydrophilic polymers, includingderivatives, may be those that include polymers in which the repeatingunits contain one or more hydroxy groups (polyhydroxy polymers),including, for example, poly(vinyl alcohol); polymers in which therepeating units contain one or more amino groups (polyamine polymers),including, for example, peptides, polypeptides, proteins andlipoproteins, such as albumin and natural lipoproteins; polymers inwhich the repeating units contain one or more carboxy groups(polycarboxy polymers), including, for example, carboxymethylcellulose,alginic acid and salts thereof, such as sodium and calcium alginate,glycosaminoglycans and salts thereof, including salts of hyaluronicacid, phosphorylated and sulfonated derivatives of carbohydrates,genetic material, such as interleukin-2 and interferon, andphosphorothioate oligomers; and polymers in which the repeating unitscontain one or more saccharide moieties (polysaccharide polymers),including, for example, carbohydrates.

The molecular weight of the hydrophilic polymers may vary, and isgenerally about 50 to about 5,000,000, with polymers having a molecularweight of about 100 to about 50,000 being preferred. The polymers may bebranched or unbranched. More preferred polymers have a molecular weightof about 150 to about 10,000, with molecular weights of 200 to about8,000 being even more preferred.

For polyethylene glycol, the preferred molecular weight is between about1 kDa and about 100 kDa (the term “about” indicating that inpreparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

Additional preferred polymers which may be used to derivatizepolypeptides of the invention, include, for example, poly(ethyleneglycol) (PEG), poly(vinylpyrrolidine), polyoxomers, polysorbate andpoly(vinyl alcohol), with PEG polymers being particularly preferred.Preferred among the PEG polymers are PEG polymers having a molecularweight of from about 100 to about 10,000. More preferably, the PEGpolymers have a molecular weight of from about 200 to about 8,000, withPEG 2,000, PEG 5,000 and PEG 8,000, which have molecular weights of2,000, 5,000 and 8,000, respectively, being even more preferred. Othersuitable hydrophilic polymers, in addition to those exemplified above,will be readily apparent to one skilled in the art based on the presentdisclosure. Generally, the polymers used may include polymers that canbe attached to the polypeptides of the invention via alkylation oracylation reactions.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (polypeptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminus) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

As with the various polymers exemplified above, it is contemplated thatthe polymeric residues may contain functional groups in addition, forexample, to those typically involved in linking the polymeric residuesto the polypeptides of the present invention. Such functionalitiesinclude, for example, carboxyl, amine, hydroxy and thiol groups. Thesefunctional groups on the polymeric residues can be further reacted, ifdesired, with materials that are generally reactive with such functionalgroups and which can assist in targeting specific tissues in the bodyincluding, for example, diseased tissue. Exemplary materials which canbe reacted with the additional functional groups include, for example,proteins, including antibodies, carbohydrates, peptides, glycopeptides,glycolipids, lectins, and nucleosides.

In addition to residues of hydrophilic polymers, the chemical used toderivatize the polypeptides of the present invention can be a saccharideresidue. Exemplary saccharides which can be derived include, forexample, monosaccharides or sugar alcohols, such as erythrose, threose,ribose, arabinose, xylose, lyxose, fructose, sorbitol, mannitol andsedoheptulose, with preferred monosaccharides being fructose, mannose,xylose, arabinose, mannitol and sorbitol; and disaccharides, such aslactose, sucrose, maltose and cellobiose. Other saccharides include, forexample, inositol and ganglioside head groups. Other suitablesaccharides, in addition to those exemplified above, will be readilyapparent to one skilled in the art based on the present disclosure.Generally, saccharides which may be used for derivitization includesaccharides that can be attached to the polypeptides of the inventionvia alkylation or acylation reactions.

Moreover, the invention also encompasses derivitization of thepolypeptides of the present invention, for example, with lipids(including cationic, anionic, polymerized, charged, synthetic,saturated, unsaturated, and any combination of the above, etc.).stabilizing agents.

The invention encompasses derivitization of the polypeptides of thepresent invention, for example, with compounds that may serve astabilizing function (e.g., to increase the polypeptides half-life insolution, to make the polypeptides more water soluble, to increase thepolypeptides hydrophilic or hydrophobic character, etc.). Polymersuseful as stabilizing materials may be of natural, semi-synthetic(modified natural) or synthetic origin. Exemplary natural polymersinclude naturally occurring polysaccharides, such as, for example,arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans,xylans (such as, for example, inulin), levan, fucoidan, carrageenan,galatocarolose, pectic acid, pectins, including amylose, pullulan,glycogen, amylopectin, cellulose, dextran, dextrin, dextrose, glucose,polyglucose, polydextrose, pustulan, chitin, agarose, keratin,chondroitin, dermatan, hyaluronic acid, alginic acid, xanthin gum,starch and various other natural homopolymer or heteropolymers, such asthose containing one or more of the following aldoses, ketoses, acids oramines: erythose, threose, ribose, arabinose, xylose, lyxose, allose,altrose, glucose, dextrose, mannose, gulose, idose, galactose, talose,erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose,mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose,glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine,aspartic acid, glutamic acid, lysine, arginine, histidine, glucuronicacid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid,glucosamine, galactosamine, and neuraminic acid, and naturally occurringderivatives thereof Accordingly, suitable polymers include, for example,proteins, such as albumin, polyalginates, and polylactide-coglycolidepolymers. Exemplary semi-synthetic polymers includecarboxymethylcellulose, hydroxymethylcellulose,hydroxypropylmethylcellulose, methylcellulose, and methoxycellulose.Exemplary synthetic polymers include polyphosphazenes, hydroxyapatites,fluoroapatite polymers, polyethylenes (such as, for example,polyethylene glycol (including for example, the class of compoundsreferred to as Pluronics.RTM., commercially available from BASF,Parsippany, N.J.), polyoxyethylene, and polyethylene terephthlate),polypropylenes (such as, for example, polypropylene glycol),polyurethanes (such as, for example, polyvinyl alcohol (PVA), polyvinylchloride and polyvinylpyrrolidone), polyamides including nylon,polystyrene, polylactic acids, fluorinated hydrocarbon polymers,fluorinated carbon polymers (such as, for example,polytetrafluoroethylene), acrylate, methacrylate, andpolymethylmethacrylate, and derivatives thereof Methods for thepreparation of derivatized polypeptides of the invention which employpolymers as stabilizing compounds will be readily apparent to oneskilled in the art, in view of the present disclosure, when coupled withinformation known in the art, such as that described and referred to inUnger, U.S. Pat. No. 5,205,290, the disclosure of which is herebyincorporated by reference herein in its entirety.

Moreover, the invention encompasses additional modifications of thepolypeptides of the present invention. Such additional modifications areknown in the art, and are specifically provided, in addition to methodsof derivitization, etc., in U.S. Pat. No. 6,028,066, which is herebyincorporated in its entirety herein.

The polypeptides of the invention may be in monomers or multimers (i.e.,dimers, trimers, tetramers and higher multimers). Accordingly, thepresent invention relates to monomers and multimers of the polypeptidesof the invention, their preparation, and compositions (preferably,Therapeutics) containing them. In specific embodiments, the polypeptidesof the invention are monomers, dimers, trimers or tetramers. Inadditional embodiments, the multimers of the invention are at leastdimers, at least trimers, or at least tetramers.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term homomer, refers to a multimer containing onlypolypeptides corresponding to the amino acid sequence of SEQ ID NO:2,24, 54, and/or 56 or encoded by the cDNA contained in a deposited clone(including fragments, variants, splice variants, and fusion proteins,corresponding to these polypeptides as described herein). These homomersmay contain polypeptides having identical or different amino acidsequences. In a specific embodiment, a homomer of the invention is amultimer containing only polypeptides having an identical amino acidsequence. In another specific embodiment, a homomer of the invention isa multimer containing polypeptides having different amino acidsequences. In specific embodiments, the multimer of the invention is ahomodimer (e.g., containing polypeptides having identical or differentamino acid sequences) or a homotrimer (e.g., containing polypeptideshaving identical and/or different amino acid sequences). In additionalembodiments, the homomeric multimer of the invention is at least ahomodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containing oneor more heterologous polypeptides (i.e., polypeptides of differentproteins) in addition to the polypeptides of the invention. In aspecific embodiment, the multimer of the invention is a heterodimer, aheterotrimer, or a heterotetramer. In additional embodiments, theheteromeric multimer of the invention is at least a heterodimer, atleast a heterotrimer, or at least a heterotetramer.

Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when polypeptides of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the polypeptides of the invention. Suchcovalent associations may involve one or more amino acid residuescontained in the polypeptide sequence (e.g., that recited in thesequence listing, or contained in the polypeptide encoded by a depositedclone). In one instance, the covalent associations are cross-linkingbetween cysteine residues located within the polypeptide sequences whichinteract in the native (i.e., naturally occurring) polypeptide. Inanother instance, the covalent associations are the consequence ofchemical or recombinant manipulation. Alternatively, such covalentassociations may involve one or more amino acid residues contained inthe heterologous polypeptide sequence in a fusion protein of theinvention.

In one example, covalent associations are between the heterologoussequence contained in a fusion protein of the invention (see, e.g., U.S.Pat. No. 5,478,925). In a specific example, the covalent associationsare between the heterologous sequence contained in an Fc fusion proteinof the invention (as described herein). In another specific example,covalent associations of fusion proteins of the invention are betweenheterologous polypeptide sequence from another protein that is capableof forming covalently associated mulfimers, such as for example,osteoprotegerin (see, e.g., International Publication NO: WO 98/49305,the contents of which are herein incorporated by reference in itsentirety). In another embodiment, two or more polypeptides of theinvention are joined through peptide linkers. Examples include thosepeptide linkers described in U.S. Pat. No. 5,073,627 (herebyincorporated by reference). Proteins comprising multiple polypeptides ofthe invention separated by peptide linkers may be produced usingconventional recombinant DNA technology.

Another method for preparing multimer polypeptides of the inventioninvolves use of polypeptides of the invention fused to a leucine zipperor isoleucine zipper polypeptide sequence. Leucine zipper and isoleucinezipper domains are polypeptides that promote multimerization of theproteins in which they are found. Leucine zippers were originallyidentified in several DNA-binding proteins (Landschulz et al., Science240:1759, (1988)), and have since been found in a variety of differentproteins. Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize. Examples ofleucine zipper domains suitable for producing soluble multimericproteins of the invention are those described in PCT application WO94/10308, hereby incorporated by reference. Recombinant fusion proteinscomprising a polypeptide of the invention fused to a polypeptidesequence that dimerizes or trimerizes in solution are expressed insuitable host cells, and the resulting soluble multimeric fusion proteinis recovered from the culture supernatant using techniques known in theart.

Trimeric polypeptides of the invention may offer the advantage ofenhanced biological activity. Preferred leucine zipper moieties andisoleucine moieties are those that preferentially form trimers. Oneexample is a leucine zipper derived from lung surfactant protein D(SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) andin U.S. patent application Ser. No. 08/446,922, hereby incorporated byreference. Other peptides derived from naturally occurring trimericproteins may be employed in preparing trmeric polypeptides of theinvention.

In another example, proteins of the invention are associated byinteractions between Flag® polypeptide sequence contained in fusionproteins of the invention containing Flag® polypeptide sequence. In afurther embodiment, associations proteins of the invention areassociated by interactions between heterologous polypeptide sequencecontained in Flag® fusion proteins of the invention and anti-Flag®antibody.

The multimers of the invention may be generated using chemicaltechniques known in the art. For example, polypeptides desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the sequence ofthe polypeptides desired to be contained in the multimer (see, e.g.,U.S. Pat. No. 5,478,925, which is herein incorporated by reference inits entirety). Further, polypeptides of the invention may be routinelymodified by the addition of cysteine or biotin to the C terminus orN-terminus of the polypeptide and techniques known in the art may beapplied to generate multimers containing one or more of these modifiedpolypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, techniquesknown in the art may be applied to generate liposomes containing thepolypeptide components desired to be contained in the multimer of theinvention (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using geneticengineering techniques known in the art. In one embodiment, polypeptidescontained in multimers of the invention are produced recombinantly usingfusion protein technology described herein or otherwise known in the art(see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated byreference in its entirety). In a specific embodiment, polynucleotidescoding for a homodimer of the invention are generated by ligating apolynucleotide sequence encoding a polypeptide of the invention to asequence encoding a linker polypeptide and then further to a syntheticpolynucleotide encoding the translated product of the polypeptide in thereverse orientation from the original C-terminus to the N-terminus(lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety). In anotherembodiment, recombinant techniques described herein or otherwise knownin the art are applied to generate recombinant polypeptides of theinvention which contain a transmembrane domain (or hydrophobic or signalpeptide) and which can be incorporated by membrane reconstitutiontechniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety).

In addition, the polynucleotide insert of the present invention could beoperatively linked to “artificial” or chimeric promoters andtranscription factors. Specifically, the artificial promoter couldcomprise, or alternatively consist, of any combination of cis-acting DNAsequence elements that are recognized by trans-acting transcriptionfactors. Preferably, the cis acting DNA sequence elements andtrans-acting transcription factors are operable in mammals. Further, thetrans-acting transcription factors of such “artificial” promoters couldalso be “artificial” or chimeric in design themselves and could act asactivators or repressors to said “artificial” promoter.

Uses of the Polynucleotides

Each of the polynucleotides identified herein can be used in numerousways as reagents. The following description should be consideredexemplary and utilizes known techniques.

The polynucleotides of the present invention are useful for chromosomeidentification. There exists an ongoing need to identify new chromosomemarkers, since few chromosome marking reagents, based on actual sequencedata (repeat polymorphisms), are presently available. Eachpolynucleotide of the present invention can be used as a chromosomemarker.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the sequences shown in SEQ ID NO:1, 23, 53,and/or 55. Primers can be selected using computer analysis so thatprimers do not span more than one predicted exon in the genomic DNA.These primers are then used for PCR screening of somatic cell hybridscontaining individual human chromosomes. Only those hybrids containingthe human gene corresponding to the SEQ ID NO: 1, 23, 53, and/or 55 willyield an amplified fragment.

Similarly, somatic hybrids provide a rapid method of PCR mapping thepolynucleotides to particular chromosomes. Three or more clones can beassigned per day using a single thermal cycler. Moreover,sublocalization of the polynucleotides can be achieved with panels ofspecific chromosome fragments. Other gene mapping strategies that can beused include in situ hybridization, prescreening with labeledflow-sorted chromosomes, and preselection by hybridization to constructchromosome specific-cDNA libraries.

Precise chromosomal location of the polynucleotides can also be achievedusing fluorescence in situ hybridization (FISH) of a metaphasechromosomal spread. This technique uses polynucleotides as short as 500or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred. Fora review of this technique, see Verma et al., “Human Chromosomes: aManual of Basic Techniques,” Pergamon Press, New York (1988).

For chromosome mapping, the polynucleotides can be used individually (tomark a single chromosome or a single site on that chromosome) or inpanels (for marking multiple sites and/or multiple chromosomes).Preferred polynucleotides correspond to the noncoding regions of thecDNAs because the coding sequences are more likely conserved within genefamilies, thus increasing the chance of cross hybridization duringchromosomal mapping.

Once a polynucleotide has been mapped to a precise chromosomal location,the physical position of the polynucleotide can be used in linkageanalysis. Linkage analysis establishes coinheritance between achromosomal location and presentation of a particular disease. Diseasemapping data are known in the art. Assuming 1 megabase mappingresolution and one gene per 20 kb, a cDNA precisely localized to achromosomal region associated with the disease could be one of 50-500potential causative genes.

Thus, once coinheritance is established, differences in thepolynucleotide and the corresponding gene between affected andunaffected organisms can be examined. First, visible structuralalterations in the chromosomes, such as deletions or translocations, areexamined in chromosome spreads or by PCR. If no structural alterationsexist, the presence of point mutations are ascertained. Mutationsobserved in some or all affected organisms, but not in normal organisms,indicates that the mutation may cause the disease. However, completesequencing of the polypeptide and the corresponding gene from severalnormal organisms is required to distinguish the mutation from apolymorphism. If a new polymorphism is identified, this polymorphicpolypeptide can be used for further linkage analysis.

Furthermore, increased or decreased expression of the gene in affectedorganisms as compared to unaffected organisms can be assessed usingpolynucleotides of the present invention. Any of these alterations(altered expression, chromosomal rearrangement, or mutation) can be usedas a diagnostic or prognostic marker.

Thus, the invention also provides a diagnostic method useful duringdiagnosis of a disorder, involving measuring the expression level ofpolynucleotides of the present invention in cells or body fluid from anorganism and comparing the measured gene expression level with astandard level of polynucleotide expression level, whereby an increaseor decrease in the gene expression level compared to the standard isindicative of a disorder.

By “measuring the expression level of a polynucleotide of the presentinvention” is intended qualitatively or quantitatively measuring orestimating the level of the polypeptide of the present invention or thelevel of the mRNA encoding the polypeptide in a first biological sampleeither directly (e.g., by determining or estimating absolute proteinlevel or mRNA level) or relatively (e.g., by comparing to thepolypeptide level or mRNA level in a second biological sample).Preferably, the polypeptide level or mRNA level in the first biologicalsample is measured or estimated and compared to a standard polypeptidelevel or mRNA level, the standard being taken from a second biologicalsample obtained from an individual not having the disorder or beingdetermined by averaging levels from a population of organisms not havinga disorder. As will be appreciated in the art, once a standardpolypeptide level or mRNA level is known, it can be used repeatedly as astandard for comparison.

By “biological sample” is intended any biological sample obtained froman organism, body fluids, cell line, tissue culture, or other sourcewhich contains the polypeptide of the present invention or mRNA. Asindicated, biological samples include body fluids (such as the followingnon-limiting examples, sputum, amniotic fluid, urine, saliva, breastmilk, secretions, interstitial fluid, blood, serum, spinal fluid, etc.)which contain the polypeptide of the present invention, and other tissuesources found to express the polypeptide of the present invention.Methods for obtaining tissue biopsies and body fluids from organisms arewell known in the art. Where the biological sample is to include mRNA, atissue biopsy is the preferred source.

The method(s) provided above may Preferably be applied in a diagnosticmethod and/or kits in which polynucleotides and/or polypeptides areattached to a solid support. In one exemplary method, the support may bea “gene chip” or a “biological chip” as described in U.S. Pat. Nos.5,837,832, 5,874,219, and 5,856,174. Further, such a gene chip withpolynucleotides of the present invention attached may be used toidentify polymorphisms between the polynucleotide sequences, withpolynucleotides isolated from a test subject. The knowledge of suchpolymorphisms (i.e. their location, as well as, their existence) wouldbe beneficial in identifying disease loci for many disorders, includingproliferative diseases and conditions. Such a method is described inU.S. Pat. Nos. 5,858,659 and 5,856,104. The US Patents referenced supraare hereby incorporated by reference in their entirety herein.

The present invention encompasses polynucleotides of the presentinvention that are chemically synthesized, or reproduced as peptidenucleic acids (PNA), or according to other methods known in the art. Theuse of PNAs would serve as the preferred form if the polynucleotides areincorporated onto a solid support, or gene chip. For the purposes of thepresent invention, a peptide nucleic acid (PNA) is a polyamide type ofDNA analog and the monomeric units for adenine, guanine, thymine andcytosine are available commercially (Perceptive Biosystems). Certaincomponents of DNA, such as phosphorus, phosphorus oxides, or deoxyribosederivatives, are not present in PNAs. As disclosed by P. E. Nielsen, M.Egholm, R. H. Berg and 0. Buchardt, Science 254, 1497 (1991); and M.Egholm, O. Buchardt, L.Christensen, C. Behrens, S. M. Freier, D. A.Driver, R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen, Nature 365,666 (1993), PNAs bind specifically and tightly to complementary DNAstrands and are not degraded by nucleases. In fact, PNA binds morestrongly to DNA than DNA itself does. This is probably because there isno electrostatic repulsion between the two strands, and also thepolyamide backbone is more flexible. Because of this, PNA/DNA duplexesbind under a wider range of stringency conditions than DNA/DNA duplexes,making it easier to perform multiplex hybridization. Smaller probes canbe used than with DNA due to the stronger binding characteristics ofPNA:DNA hybrids. In addition, it is more likely that single basemismatches can be determined with PNA/DNA hybridization because a singlemismatch in a PNA/DNA 15-mer lowers the melting point (T.sub.m) by8°-20° C., vs. 4°-16° C. for the DNA/DNA 15-mer duplex. Also, theabsence of charge groups in PNA means that hybridization can be done atlow ionic strengths and reduce possible interference by salt during theanalysis.

In addition to the foregoing, a polynucleotide can be used to controlgene expression through triple helix formation or antisense DNA or RNA.Antisense techniques are discussed, for example, in Okano, J. Neurochem.56: 560 (1991); “Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance Lee et al., Nucleic Acids Research 6: 3073(1979); Cooney et al., Science 241: 456 (1988); and Dervan et al.,Science 251: 1360 (1991). Both methods rely on binding of thepolynucleotide to a complementary DNA or RNA. For these techniques,preferred polynucleotides are usually oligonucleotides 20 to 40 bases inlength and complementary to either the region of the gene involved intranscription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073(1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1360 (1991) ) or to the mRNA itself (antisense—Okano, J.Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988).) Triple helixformation optimally results in a shut-off of RNA transcription from DNA,while antisense RNA hybridization blocks translation of an niRNAmolecule into polypeptide. Both techniques are effective in modelsystems, and the information disclosed herein can be used to designantisense or triple helix polynucleotides in an effort to treat orprevent disease.

The present invention encompasses the addition of a nuclear localizationsignal, operably linked to the 5′ end, 3′ end, or any location therein,to any of the oligonucleotides, antisense oligonucleotides, triple helixoligonucleotides, ribozymes, PNA oligonucleotides, and/orpolynucleotides, of the present invention. See, for example, G. Cutrona,et al., Nat. Biotech., 18:300-303, (2000); which is hereby incorporatedherein by reference.

Polynucleotides of the present invention are also useful in genetherapy. One goal of gene therapy is to insert a normal gene into anorganism having a defective gene, in an effort to correct the geneticdefect. The polynucleotides disclosed in the present invention offer ameans of targeting such genetic defects in a highly accurate manner.Another goal is to insert a new gene that was not present in the hostgenome, thereby producing a new trait in the host cell. In one example,polynucleotide sequences of the present invention may be used toconstruct chimeric RNA/DNA oligonucleotides corresponding to saidsequences, specifically designed to induce host cell mismatch repairmechanisms in an organism upon systemic injection, for example(Bartlett, R. J., et al., Nat. Biotech, 18:615-622 (2000), which ishereby incorporated by reference herein in its entirety). Such RNA/DNAoligonucleotides could be designed to correct genetic defects in certainhost strains, and/or to introduce desired phenotypes in the host (e.g.,introduction of a specific polymorphism within an endogenous genecorresponding to a polynucleotide of the present invention that mayameliorate and/or prevent a disease symptom and/or disorder, etc.).Alternatively, the polynucleotide sequence of the present invention maybe used to construct duplex oligonucleotides corresponding to saidsequence, specifically designed to correct genetic defects in certainhost strains, and/or to introduce desired phenotypes into the host(e.g., introduction of a specific polymorphism within an endogenous genecorresponding to a polynucleotide of the present invention that mayameliorate and/or prevent a disease symptom and/or disorder, etc). Suchmethods of using duplex oligonucleotides are known in the art and areencompassed by the present invention (see EP1007712, which is herebyincorporated by reference herein in its entirety).

The polynucleotides are also useful for identifying organisms fromminute biological samples. The United States military, for example, isconsidering the use of restriction fragment length polymorphism (RFLP)for identification of its personnel. In this technique, an individual'sgenomic DNA is digested with one or more restriction enzymes, and probedon a Southern blot to yield unique bands for identifying personnel. Thismethod does not suffer from the current limitations of “Dog Tags” whichcan be lost, switched, or stolen, making positive identificationdifficult. The polynucleotides of the present invention can be used asadditional DNA markers for RFLP.

The polynucleotides of the present invention can also be used as analternative to RFLP, by determining the actual base-by-base DNA sequenceof selected portions of an organisms genome. These sequences can be usedto prepare PCR primers for amplifiing and isolating such selected DNA,which can then be sequenced. Using this technique, organisms can beidentified because each organism will have a unique set of DNAsequences. Once an unique ID database is established for an organism,positive identification of that organism, living or dead, can be madefrom extremely small tissue samples. Similarly, polynucleotides of thepresent invention can be used as polymorphic markers, in addition to,the identification of transformed or non-transformed cells and/ortissues.

There is also a need for reagents capable of identifying the source of aparticular tissue. Such need arises, for example, when presented withtissue of unknown origin. Appropriate reagents can comprise, forexample, DNA probes or primers specific to particular tissue preparedfrom the sequences of the present invention. Panels of such reagents canidentify tissue by species and/or by organ type. In a similar fashion,these reagents can be used to screen tissue cultures for contamination.Moreover, as mentioned above, such reagents can be used to screen and/oridentify transformed and non-transformed cells and/or tissues.

In the very least, the polynucleotides of the present invention can beused as molecular weight markers on Southern gels, as diagnostic probesfor the presence of a specific mRNA in a particular cell type, as aprobe to “subtract-out” known sequences in the process of discoveringnovel polynucleotides, for selecting and making oligomers for attachmentto a “gene chip” or other support, to raise anti-DNA antibodies usingDNA immunization techniques, and as an antigen to elicit an immuneresponse.

Uses of the Polypeptides

Each of the polypeptides identified herein can be used in numerous ways.The following description should be considered exemplary and utilizesknown techniques.

A polypeptide of the present invention can be used to assay proteinlevels in a biological sample using antibody-based techniques. Forexample, protein expression in tissues can be studied with classicalimmunohistological methods. (Jalkanen, M., et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096(1987).) Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase, and radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (1 12In), and technetium (99mTc), andfluorescent labels, such as fluorescein and rhodamine, and biotin.

In addition to assaying protein levels in a biological sample, proteinscan also be detected in vivo by imaging. Antibody labels or markers forin vivo imaging of protein include those detectable by X-radiography,NoM or ESR. For X-radiography, suitable labels include radioisotopessuch as barium or cesium, which emit detectable radiation but are notovertly harmful to the subject. Suitable markers for NMR and ESR includethose with a detectable characteristic spin, such as deuterium, whichmay be incorporated into the antibody by labeling of nutrients for therelevant hybridoma.

A protein-specific antibody or antibody fragment which has been labeledwith an appropriate detectable imaging moiety, such as a radioisotope(for example, 131I, 112In, 99mTc), a radio-opaque substance, or amaterial detectable by nuclear magnetic resonance, is introduced (forexample, parenterally, subcutaneously, or intraperitoneally) into themammal. It will be understood in the art that the size of the subjectand the imaging system used will determine the quantity of imagingmoiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of 99mTc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain the specific protein.In vivo tumor imaging is described in S. W. Burchiel et al.,“Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.”(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).)

Thus, the invention provides a diagnostic method of a disorder, whichinvolves (a) assaying the expression of a polypeptide of the presentinvention in cells or body fluid of an individual; (b) comparing thelevel of gene expression with a standard gene expression level, wherebyan increase or decrease in the assayed polypeptide gene expression levelcompared to the standard expression level is indicative of a disorder.With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Moreover, polypeptides of the present invention can be used to treat,prevent, and/or diagnose disease. For example, patients can beadministered a polypeptide of the present invention in an effort toreplace absent or decreased levels of the polypeptide (e.g., insulin),to supplement absent or decreased levels of a different polypeptide(e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repairproteins), to inhibit the activity of a polypeptide (e.g., an oncogeneor tumor suppressor), to activate the activity of a polypeptide (e.g.,by binding to a receptor), to reduce the activity of a membrane boundreceptor by competing with it for free ligand (e.g., soluble TNFreceptors used in reducing inflammation), or to bring about a desiredresponse (e.g., blood vessel growth inhibition, enhancement of theimmune response to proliferative cells or tissues).

Similarly, antibodies directed to a polypeptide of the present inventioncan also be used to treat, prevent, and/or diagnose disease. Forexample, administration of an antibody directed to a polypeptide of thepresent invention can bind and reduce overproduction of the polypeptide.Similarly, administration of an antibody can activate the polypeptide,such as by binding to a polypeptide bound to a membrane (receptor).

At the very least, the polypeptides of the present invention can be usedas molecular weight markers on SDS-PAGE gels or on molecular sieve gelfiltration columns using methods well known to those of skill in theart. Polypeptides can also be used to raise antibodies, which in turnare used to measure protein expression from a recombinant cell, as a wayof assessing transformation of the host cell. Moreover, the polypeptidesof the present invention can be used to test the following biologicalactivities.

Gene Therapy Methods

Another aspect of the present invention is to gene therapy methods fortreating or preventing disorders, diseases and conditions. The genetherapy methods relate to the introduction of nucleic acid (DNA, RNA andantisense DNA or RNA) sequences into an animal to achieve expression ofa polypeptide of the present invention. This method requires apolynucleotide which codes for a polypeptide of the invention thatoperatively linked to a promoter and any other genetic elementsnecessary for the expression of the polypeptide by the target tissue.Such gene therapy and delivery techniques are known in the art, see, forexample, WO90/11092, which is herein incorporated by reference.

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) comprising a promoter operably linked to apolynucleotide of the invention ex vivo, with the engineered cells thenbeing provided to a patient to be treated with the polypeptide. Suchmethods are well-known in the art. For example, see Belldegrun et al.,J. Natl. Cancer Inst., 85:207-216 (1993); Ferrantini et al., CancerResearch, 53:107-1112 (1993); Ferrantini et al., J. Immunology 153:46044615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995);Ogura et al., Cancer Research 50: 5102-5106 (1990); Santodonato, et al.,Human Gene Therapy 7:1-10 (1996); Santodonato, et al., Gene Therapy4:1246-1255 (1997); and Zhang, et al., Cancer Gene Therapy 3: 31-38(1996)), which are herein incorporated by reference. In one embodiment,the cells which are engineered are arterial cells. The arterial cellsmay be reintroduced into the patient through direct injection to theartery, the tissues surrounding the artery, or through catheterinjection.

As discussed in more detail below, the polynucleotide constructs can bedelivered by any method that delivers injectable materials to the cellsof an animal, such as, injection into the interstitial space of tissues(heart, muscle, skin, lung, liver, and the like). The polynucleotideconstructs may be delivered in a pharmaceutically acceptable liquid oraqueous carrier.

In one embodiment, the polynucleotide of the invention is delivered as anaked polynucleotide. The term “naked” polynucleotide, DNA or RNA refersto sequences that are free from any delivery vehicle that acts toassist, promote or facilitate entry into the cell, including viralsequences, viral particles, liposome formulations, lipofectin orprecipitating agents and the like. However, the polynucleotides of theinvention can also be delivered in liposome formulations and lipofectinformulations and the like can be prepared by methods well known to thoseskilled in the art. Such methods are described, for example, in U.S.Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are hereinincorporated by reference.

The polynucleotide vector constructs of the invention used in the genetherapy method are preferably constructs that will not integrate intothe host genome nor will they contain sequences that allow forreplication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL availablefrom Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available fromInvitrogen. Other suitable vectors will be readily apparent to theskilled artisan.

Any strong promoter known to those skilled in the art can be used fordriving the expression of polynucleotide sequence of the invention.Suitable promoters include adenoviral promoters, such as the adenoviralmajor late promoter; or heterologous promoters, such as thecytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV)promoter; inducible promoters, such as the MMT promoter, themetallothionein promoter; heat shock promoters; the albumin promoter;the ApoAl promoter; human globin promoters; viral thymidine kinasepromoters, such as the Herpes Simplex thymidine kinase promoter;retroviral LTRs; the b-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter for thepolynucleotides of the invention.

Unlike other gene therapy techniques, one major advantage of introducingnaked nucleic acid sequences into target cells is the transitory natureof the polynucleotide synthesis in the cells. Studies have shown thatnon-replicating DNA sequences can be introduced into cells to provideproduction of the desired polypeptide for periods of up to six months.

The polynucleotide construct of the invention can be delivered to theinterstitial space of tissues within the an animal, including of muscle,skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph,blood, bone, cartilage, pancreas, kidney, gall bladder, stomach,intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,and connective tissue. Interstitial space of the tissues comprises theintercellular, fluid, mucopolysaccharide matrix among the reticularfibers of organ tissues, elastic fibers in the walls of vessels orchambers, collagen fibers of fibrous tissues, or that same matrix withinconnective tissue ensheathing muscle cells or in the lacunae of bone. Itis similarly the space occupied by the plasma of the circulation and thelymph fluid of the lymphatic channels. Delivery to the interstitialspace of muscle tissue is preferred for the reasons discussed below.They may be conveniently delivered by injection into the tissuescomprising these cells. They are preferably delivered to and expressedin persistent, non-dividing cells which are differentiated, althoughdelivery and expression may be achieved in non-differentiated or lesscompletely differentiated cells, such as, for example, stem cells ofblood or skin fibroblasts. In vivo muscle cells are particularlycompetent in their ability to take up and express polynucleotides.

For the naked nucleic acid sequence injection, an effective dosageamount of DNA or RNA will be in the range of from about 0.05 mg/kg bodyweight to about 50 mg/kg body weight. Preferably the dosage will be fromabout 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.

The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked DNAconstructs can be delivered to arteries during angioplasty by thecatheter used in the procedure.

The naked polynucleotides are delivered by any method known in the art,including, but not limited to, direct needle injection at the deliverysite, intravenous injection, topical administration, catheter infusion,and so-called “gene guns”. These delivery methods are known in the art.

The constructs may also be delivered with delivery vehicles such asviral sequences, viral particles, liposome formulations, lipofectin,precipitating agents, etc. Such methods of delivery are known in theart.

In certain embodiments, the polynucleotide constructs of the inventionare complexed in a liposome preparation. Liposomal preparations for usein the instant invention include cationic (positively charged), anionic(negatively charged) and neutral preparations. However, cationicliposomes are particularly preferred because a tight charge complex canbe formed between the cationic liposome and the polyanionic nucleicacid. Cationic liposomes have been shown to mediate intracellulardelivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA ,84:7413-7416 (1987), which is herein incorporated by reference); mRNA(Malone et al., Proc. Natl. Acad. Sci. USA, 86:6077-6081 (1989), whichis herein incorporated by reference); and purified transcription factors(Debs et al., J. Biol. Chem., 265:10189-10192 (1990), which is hereinincorporated by reference), in functional form.

Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areparticularly useful and are available under the trademark Lipofectin,from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc.Natl. Acad. Sci. USA , 84:7413-7416 (1987), which is herein incorporatedby reference). Other commercially available liposomes includetransfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

Other cationic liposomes can be prepared from readily availablematerials using techniques well known in the art. See, e.g. PCTPublication NO: WO 90/11092 (which is herein incorporated by reference)for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparationof DOTMA liposomes is explained in the literature, see, e.g., Felgner etal., Proc. Natl. Acad. Sci. USA, 84:7413-7417, which is hereinincorporated by reference. Similar methods can be used to prepareliposomes from other cationic lipid materials.

Similarly, anionic and neutral liposomes are readily available, such asfrom Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidyl,choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

For example, commercially dioleoylphosphatidyl choline (DOPC),dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15EC. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

The liposomes can comprise multilamellar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), withSUVs being preferred. The various liposome-nucleic acid complexes areprepared using methods well known in the art. See, e.g., Straubinger etal., Methods of Immunology, 101:512-527 (1983), which is hereinincorporated by reference. For example, MLVs containing nucleic acid canbe prepared by depositing a thin film of phospholipid on the walls of aglass tube and subsequently hydrating with a solution of the material tobe encapsulated. SUVs are prepared by extended sonication of MLVs toproduce a homogeneous population of unilamellar liposomes. The materialto be entrapped is added to a suspension of preformed MLVs and thensonicated. When using liposomes containing cationic lipids, the driedlipid film is resuspended in an appropriate solution such as sterilewater or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated,and then the preformed liposomes are mixed directly with the DNA. Theliposome and DNA form a very stable complex due to binding of thepositively charged liposomes to the cationic DNA. SUVs find use withsmall nucleic acid fragments. LUVs are prepared by a number of methods,well known in the art. Commonly used methods include Ca2+-EDTA chelation(Papahadjopoulos et al., Biochim. Biophys. Acta, 394:483 (1975); Wilsonet al., Cell, 17:77 (1979)); ether injection (Deamer et al., Biochim.Biophys. Acta, 443:629 (1976); Ostro et al., Biochem. Biophys. Res.Commun., 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA,76:3348 (1979)); detergent dialysis (Enoch et al., Proc. Natl. Acad.Sci. USA , 76:145 (1979)); and reverse-phase evaporation (REV) (Fraleyet al., J. Biol. Chem., 255:10431 (1980); Szoka et al., Proc. Natl.Acad. Sci. USA, 75:145 (1978); Schaefer-Ridder et al., Science, 215:166(1982)), which are herein incorporated by reference.

Generally, the ratio of DNA to liposomes will be from about 10:1 toabout 1:10. Preferably, the ration will be from about 5:1 to about 1:5.More preferably, the ration will be about 3:1 to about 1:3. Still morepreferably, the ratio will be about 1:1.

U.S. Pat. No.: 5,676,954 (which is herein incorporated by reference)reports on the injection of genetic material, complexed with cationicliposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787,5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, andinternational publication NO: WO 94/9469 (which are herein incorporatedby reference) provide cationic lipids for use in transfecting DNA intocells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication NO: WO 94/9469 (which areherein incorporated by reference) provide methods for deliveringDNA-cationic lipid complexes to mammals.

In certain embodiments, cells are engineered, ex vivo or in vivo, usinga retroviral particle containing RNA which comprises a sequence encodingpolypeptides of the invention. Retroviruses from which the retroviralplasmid vectors may be derived include, but are not limited to, MoloneyMurine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, HarveySarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, humanimmunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammarytumor virus.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, R-2,R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAml2, andDAN cell lines as described in Miller, Human Gene Therapy, 1:5-14(1990), which is incorporated herein by reference in its entirety. Thevector may transduce the packaging cells through any means known in theart. Such means include, but are not limited to, electroporation, theuse of liposomes, and CaPO4 precipitation. In one alternative, theretroviral plasmid vector may be encapsulated into a liposome, orcoupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include polynucleotide encoding polypeptides of the invention.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express polypeptides of the invention.

In certain other embodiments, cells are engineered, ex vivo or in vivo,with polynucleotides of the invention contained in an adenovirus vector.Adenovirus can be manipulated such that it encodes and expressespolypeptides of the invention, and at the same time is inactivated interms of its ability to replicate in a normal lytic viral life cycle.Adenovirus expression is achieved without integration of the viral DNAinto the host cell chromosome, thereby alleviating concerns aboutinsertional mutagenesis. Furthermore, adenoviruses have been used aslive enteric vaccines for many years with an excellent safety profile(Schwartzet al., Am. Rev. Respir. Dis., 109:233-238 (1974)). Finally,adenovirus mediated gene transfer has been demonstrated in a number ofinstances including transfer of alpha-I-antitrypsin and CFTR to thelungs of cotton rats (Rosenfeld et al., Science, 252:431-434 (1991);Rosenfeld et al., Cell, 68:143-155 (1992)). Furthermore, extensivestudies to attempt to establish adenovirus as a causative agent in humancancer were uniformly negative (Green et al. Proc. Natl. Acad. Sci. USA,76:6606 (1979)).

Suitable adenoviral vectors useful in the present invention aredescribed, for example, in Kozarsky and Wilson, Curr. Opin. Genet.Devel., 3:499-503 (1993); Rosenfeld et al., Cell, 68:143-155 (1992);Engelhardt et al., Human Genet. Ther., 4:759-769 (1993); Yang et al.,Nature Genet., 7:362-369 (1994); Wilson et al., Nature, 365:691-692(1993); and U.S. Pat. No.: 5,652,224, which are herein incorporated byreference. For example, the adenovirus vector Ad2 is useful and can begrown in human 293 cells. These cells contain the E1 region ofadenovirus and constitutively express E1a and E1b, which complement thedefective adenoviruses by providing the products of the genes deletedfrom the vector. In addition to Ad2, other varieties of adenovirus(e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

Preferably, the adenoviruses used in the present invention arereplication deficient. Replication deficient adenoviruses require theaid of a helper virus and/or packaging cell line to form infectiousparticles. The resulting virus is capable of infecting cells and canexpress a polynucleotide of interest which is operably linked to apromoter, but cannot replicate in most cells. Replication deficientadenoviruses may be deleted in one or more of all or a portion of thefollowing genes: E1a, E1b, E3, E4, E2a, or L1 through L5.

In certain other embodiments, the cells are engineered, ex vivo or invivo, using an adeno-associated virus (AAV). AAVs are naturallyoccurring defective viruses that require helper viruses to produceinfectious particles (Muzyczka, Curr. Topics in Microbiol. Immunol.,158:97 (1992)). It is also one of the few viruses that may integrate itsDNA into non-dividing cells. Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate, but space for exogenousDNA is limited to about 4.5 kb. Methods for producing and using suchAAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941,5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

For example, an appropriate AAV vector for use in the present inventionwill include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The polynucleotide constructcontaining polynucleotides of the invention is inserted into the AAVvector using standard cloning methods, such as those found in Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press(1989). The recombinant AAV vector is then transfected into packagingcells which are infected with a helper virus, using any standardtechnique, including lipofection, electroporation, calcium phosphateprecipitation, etc. Appropriate helper viruses include adenoviruses,cytomegaloviruses, vaccinia viruses, or herpes viruses. Once thepackaging cells are transfected and infected, they will produceinfectious AAV viral particles which contain the polynucleotideconstruct of the invention. These viral particles are then used totransduce eukaryotic cells, either ex vivo or in vivo. The transducedcells will contain the polynucleotide construct integrated into itsgenome, and will express the desired gene product.

Another method of gene therapy involves operably associatingheterologous control regions and endogenous polynucleotide sequences(e.g. encoding the polypeptide sequence of interest) via homologousrecombination (see, e.g., U.S. Pat. No.: 5,641,670, issued Jun. 24,1997; International Publication NO: WO 96/29411, published Sep. 26,1996; International Publication NO: WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); andZijlstra et al., Nature, 342:435-438 (1989). This method involves theactivation of a gene which is present in the target cells, but which isnot normally expressed in the cells, or is expressed at a lower levelthan desired.

Polynucleotide constructs are made, using standard techniques known inthe art, which contain the promoter with targeting sequences flankingthe promoter. Suitable promoters are described herein. The targetingsequence is sufficiently complementary to an endogenous sequence topermit homologous recombination of the promoter-targeting sequence withthe endogenous sequence. The targeting sequence will be sufficientlynear the 5′ end of the desired endogenous polynucleotide sequence so thepromoter will be operably linked to the endogenous sequence uponhomologous recombination.

The promoter and the targeting sequences can be amplified using PCR.Preferably, the amplified promoter contains distinct restriction enzymesites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 5′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3′ end of theamplified promoter. The amplified promoter and targeting sequences aredigested and ligated together.

The promoter-targeting sequence construct is delivered to the cells,either as naked polynucleotide, or in conjunction withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, whole viruses, lipofection, precipitating agents, etc.,described in more detail above. The P promoter-targeting sequence can bedelivered by any method, included direct needle injection, intravenousinjection, topical administration, catheter infusion, particleaccelerators, etc. The methods are described in more detail below.

The promoter-targeting sequence construct is taken up by cells.Homologous recombination between the construct and the endogenoussequence takes place, such that an endogenous sequence is placed underthe control of the promoter. The promoter then drives the expression ofthe endogenous sequence.

The polynucleotides encoding polypeptides of the present invention maybe administered along with other polynucleotides encoding angiogenicproteins. Angiogenic proteins include, but are not limited to, acidicand basic fibroblast growth factors, VEGF-1, VEGF-2 (VEGF-C), VEGF-3(VEGF-B), epidermal growth factor alpha and beta, platelet-derivedendothelial cell growth factor, platelet-derived growth factor, tumornecrosis factor alpha, hepatocyte growth factor, insulin like growthfactor, colony stimulating factor, macrophage colony stimulating factor,granulocyte/macrophage colony stimulating factor, and nitric oxidesynthase.

Preferably, the polynucleotide encoding a polypeptide of the inventioncontains a secretory signal sequence that facilitates secretion of theprotein. Typically, the signal sequence is positioned in the codingregion of the polynucleotide to be expressed towards or at the 5′ end ofthe coding region. The signal sequence may be homologous or heterologousto the polynucleotide of interest and may be homologous or heterologousto the cells to be transfected. Additionally, the signal sequence may bechemically synthesized using methods known in the art.

Any mode of administration of any of the above-described polynucleotidesconstructs can be used so long as the mode results in the expression ofone or more molecules in an amount sufficient to provide a therapeuticeffect. This includes direct needle injection, systemic injection,catheter infusion, biolistic injectors, particle accelerators (i.e.,“gene guns”), gelfoam sponge depots, other commercially available depotmaterials, osmotic pumps (e.g., Alza minipumps), oral or suppositorialsolid (tablet or pill) pharmaceutical formulations, and decanting ortopical applications during surgery. For example, direct injection ofnaked calcium phosphate-precipitated plasmid into rat liver and ratspleen or a protein-coated plasmid into the portal vein has resulted ingene expression of the foreign gene in the rat livers. (Kaneda et al.,Science, 243:375 (1989)).

A preferred method of local administration is by direct injection.Preferably, a recombinant molecule of the present invention complexedwith a delivery vehicle is administered by direct injection into orlocally within the area of arteries. Administration of a compositionlocally within the area of arteries refers to injecting the compositioncentimeters and preferably, millimeters within arteries.

Another method of local administration is to contact a polynucleotideconstruct of the present invention in or around a surgical wound. Forexample, a patient can undergo surgery and the polynucleotide constructcan be coated on the surface of tissue inside the wound or the constructcan be injected into areas of tissue inside the wound.

Therapeutic compositions useful in systemic administration, includerecombinant molecules of the present invention complexed to a targeteddelivery vehicle of the present invention. Suitable delivery vehiclesfor use with systemic administration comprise liposomes comprisingligands for targeting the vehicle to a particular site.

Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA, 189:11277-11281 (1992), which is incorporated herein by reference).Oral delivery can be performed by complexing a polynucleotide constructof the present invention to a carrier capable of withstandingdegradation by digestive enzymes in the gut of an animal. Examples ofsuch carriers, include plastic capsules or tablets, such as those knownin the art. Topical delivery can be performed by mixing a polynucleotideconstruct of the present invention with a lipophilic reagent (e.g.,DMSO) that is capable of passing into the skin.

Determining an effective amount of substance to be delivered can dependupon a number of factors including, for example, the chemical structureand biological activity of the substance, the age and weight of theanimal, the precise condition requiring treatment and its severity, andthe route of administration. The frequency of treatments depends upon anumber of factors, such as the amount of polynucleotide constructsadministered per dose, as well as the health and history of the subject.The precise amount, number of doses, and timing of doses will bedetermined by the attending physician or veterinarian. Therapeuticcompositions of the present invention can be administered to any animal,preferably to mammals and birds. Preferred mammals include humans, dogs,cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humansbeing particularly preferred.

Biological Activities

The polynucleotides or polypeptides, or agonists or antagonists of thepresent invention can be used in assays to test for one or morebiological activities. If these polynucleotides and polypeptides doexhibit activity in a particular assay, it is likely that thesemolecules may be involved in the diseases associated with the biologicalactivity. Thus, the polynucleotides or polypeptides, or agonists orantagonists could be used to treat the associated disease.

Immune Activity

The polynucleotides or polypeptides, or agonists or antagonists of thepresent invention may be useful in treating, preventing, and/ordiagnosing diseases, disorders, and/or conditions of the immune system,by activating or inhibiting the proliferation, differentiation, ormobilization (chemotaxis) of immune cells. Immune cells develop througha process called hematopoiesis, producing myeloid (platelets, red bloodcells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes)cells from pluripotent stem cells. The etiology of these immunediseases, disorders, and/or conditions may be genetic, somatic, such ascancer or some autoimmune diseases, disorders, and/or conditions,acquired (e.g., by chemotherapy or toxins), or infectious. Moreover, apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention can be used as a marker or detector of a particularimmune system disease or disorder.

A polynucleotides or polypeptides, or agonists or antagonists of thepresent invention may be useful in treating, preventing, and/ordiagnosing diseases, disorders, and/or conditions of hematopoieticcells. A polynucleotides or polypeptides, or agonists or antagonists ofthe present invention could be used to increase differentiation andproliferation of hematopoietic cells, including the pluripotent stemcells, in an effort to treat or prevent those diseases, disorders,and/or conditions associated with a decrease in certain (or many) typeshematopoietic cells. Examples of immunologic deficiency syndromesinclude, but are not limited to: blood protein diseases, disorders,and/or conditions (e.g. agammaglobulinemia, dysgammaglobulinemia),ataxia telangiectasia, common variable immunodeficiency, DigeorgeSyndrome, HIV infection, HTLV-BLV infection, leukocyte adhesiondeficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction,severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder,anemia, thrombocytopenia, or hemoglobinuria.

Moreover, a polynucleotides or polypeptides, or agonists or antagonistsof the present invention could also be used to modulate hemostatic (thestopping of bleeding) or thrombolytic activity (clot formation). Forexample, by increasing hemostatic or thrombolytic activity, apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention could be used to treat or prevent blood coagulationdiseases, disorders, and/or conditions (e.g., afibrinogenemia, factordeficiencies, arterial thrombosis, venous thrombosis, etc.), bloodplatelet diseases, disorders, and/or conditions (e.g. thrombocytopenia),or wounds resulting from trauma, surgery, or other causes.Alternatively, a polynucleotides or polypeptides, or agonists orantagonists of the present invention that can decrease hemostatic orthrombolytic activity could be used to inhibit or dissolve clotting.Polynucleotides or polypeptides, or agonists or antagonists of thepresent invention are may also be useful for the detection, prognosis,treatment, and/or prevention of heart attacks (infarction), strokes,scarring, fibrinolysis, uncontrolled bleeding, uncontrolled coagulation,uncontrolled complement fixation, and/or inflammation.

A polynucleotides or polypeptides, or agonists or antagonists of thepresent invention may also be useful in treating, preventing, and/ordiagnosing autoimmune diseases, disorders, and/or conditions. Manyautoimmune diseases, disorders, and/or conditions result frominappropriate recognition of self as foreign material by immune cells.This inappropriate recognition results in an immune response leading tothe destruction of the host tissue. Therefore, the administration of apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention that inhibits an immune response, particularly theproliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing autoimmune diseases, disorders, and/orconditions.

Examples of autoimmune diseases, disorders, and/or conditions that canbe treated, prevented, and/or diagnosed or detected by the presentinvention include, but are not limited to: Addison's Disease, hemolyticanemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis,allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome,Graves' Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis,Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocrinopathies,Purpura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis,Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation,Guillain-Barre Syndrome, insulin dependent diabetes mellitis, andautoimmune inflammatory eye disease.

Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated, prevented, and/or diagnosed by polynucleotides orpolypeptides, or agonists or antagonists of the present invention.Moreover, these molecules can be used to treat anaphylaxis,hypersensitivity to an antigenic molecule, or blood groupincompatibility.

A polynucleotides or polypeptides, or agonists or antagonists of thepresent invention may also be used to treat, prevent, and/or diagnoseorgan rejection or graft-versus-host disease (GVHD). Organ rejectionoccurs by host immune cell destruction of the transplanted tissuethrough an immune response. Similarly, an immune response is alsoinvolved in GVHD, but, in this case, the foreign transplanted immunecells destroy the host tissues. The administration of a polynucleotidesor polypeptides, or agonists or antagonists of the present inventionthat inhibits an immune response, particularly the proliferation,differentiation, or chemotaxis of T-cells, may be an effective therapyin preventing organ rejection or GVHD.

Similarly, a polynucleotides or polypeptides, or agonists or antagonistsof the present invention may also be used to modulate inflammation. Forexample, the polypeptide or polynucleotide or agonists or antagonist mayinhibit the proliferation and differentiation of cells involved in aninflammatory response. These molecules can be used to treat, prevent,and/or diagnose inflammatory conditions, both chronic and acuteconditions, including chronic prostatitis, granulomatous prostatitis andmalacoplakia, inflammation associated with infection (e.g., septicshock, sepsis, or systemic inflammatory response syndrome (SIRS)),ischemia-reperfusion injury, endotoxin lethality, arthritis,complement-mediated hyperacute rejection, nephritis, cytokine orchemokine induced lung injury, inflammatory bowel disease, Crohn'sdisease, or resulting from over production of cytokines (e.g., TNF orIL-1.)

Hyperproliferative Disorders

A polynucleotides or polypeptides, or agonists or antagonists of theinvention can be used to treat, prevent, and/or diagnosehyperproliferative diseases, disorders, and/or conditions, includingneoplasms. A polynucleotides or polypeptides, or agonists or antagonistsof the present invention may inhibit the proliferation of the disorderthrough direct or indirect interactions. Alternatively, apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention may proliferate other cells which can inhibit thehyperproliferative disorder.

For example, by increasing an immune response, particularly increasingantigenic qualities of the hyperproliferative disorder or byproliferating, differentiating, or mobilizing T-cells,hyperproliferative diseases, disorders, and/or conditions can betreated, prevented, and/or diagnosed. This immune response may beincreased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, decreasing an immuneresponse may also be a method of treating, preventing, and/or diagnosinghyperproliferative diseases, disorders, and/or conditions, such as achemotherapeutic agent.

Examples of hyperproliferative diseases, disorders, and/or conditionsthat can be treated, prevented, and/or diagnosed by polynucleotides orpolypeptides, or agonists or antagonists of the present inventioninclude, but are not limited to neoplasms located in the: colon,abdomen, bone, breast, digestive system, liver, pancreas, peritoneum,endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary,thymus, thyroid), eye, head and neck, nervous (central and peripheral),lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, andurogenital.

Similarly, other hyperproliferative diseases, disorders, and/orconditions can also be treated, prevented, and/or diagnosed by apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention. Examples of such hyperproliferative diseases,disorders, and/or conditions include, but are not limited to:hypergammaglobulinemia, lymphoproliferative diseases, disorders, and/orconditions, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome,Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, andany other hyperproliferative disease, besides neoplasia, located in anorgan system listed above.

One preferred embodiment utilizes polynucleotides of the presentinvention to inhibit aberrant cellular division, by gene therapy usingthe present invention, and/or protein fusions or fragments thereof.

Thus, the present invention provides a method for treating or preventingcell proliferative diseases, disorders, and/or conditions by insertinginto an abnormally proliferating cell a polynucleotide of the presentinvention, wherein'said polynucleotide represses said expression.

Another embodiment of the present invention provides a method oftreating or preventing cell-proliferative diseases, disorders, and/orconditions in individuals comprising administration of one or moreactive gene copies of the present invention to an abnormallyproliferating cell or cells. In a preferred embodiment, polynucleotidesof the present invention is a DNA construct comprising a recombinantexpression vector effective in expressing a DNA sequence encoding saidpolynucleotides. In another preferred embodiment of the presentinvention, the DNA construct encoding the polynucleotides of the presentinvention is inserted into cells to be treated utilizing a retrovirus,or more Preferably an adenoviral vector (See G J. Nabel, et. al., PNAS1999 96: 324-326, which is hereby incorporated by reference). In a mostpreferred embodiment, the viral vector is defective and will nottransform non-proliferating cells, only proliferating cells. Moreover,in a preferred embodiment, the polynucleotides of the present inventioninserted into proliferating cells either alone, or in combination withor fused to other polynucleotides, can then be modulated via an externalstimulus (i.e. magnetic, specific small molecule, chemical, or drugadministration, etc.), which acts upon the promoter upstream of saidpolynucleotides to induce expression of the encoded protein product. Assuch the beneficial therapeutic affect of the present invention may beexpressly modulated (i.e. to increase, decrease, or inhibit expressionof the present invention) based upon said external stimulus.

Polynucleotides of the present invention may be useful in repressingexpression of oncogenic genes or antigens. By “repressing expression ofthe oncogenic genes ” is intended the suppression of the transcriptionof the gene, the degradation of the gene transcript (pre-message RNA),the inhibition of splicing, the destruction of the messenger RNA, theprevention of the post-translational modifications of the protein, thedestruction of the protein, or the inhibition of the normal function ofthe protein.

For local administration to abnormally proliferating cells,polynucleotides of the present invention may be administered by anymethod known to those of skill in the art including, but not limited totransfection, electroporation, microinjection of cells, or in vehiclessuch as liposomes, lipofectin, or as naked polynucleotides, or any othermethod described throughout the specification. The polynucleotide of thepresent invention may be delivered by known gene delivery systems suchas, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845(1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad.Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol.Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yateset al., Nature 313:812 (1985)) known to those skilled in the art. Thesereferences are exemplary only and are hereby incorporated by reference.In order to specifically deliver or transfect cells which are abnormallyproliferating and spare non-dividing cells, it is preferable to utilizea retrovirus, or adenoviral (as described in the art and elsewhereherein) delivery system known to those of skill in the art. Since hostDNA replication is required for retroviral DNA to integrate and theretrovirus will be unable to self replicate due to the lack of theretrovirus genes needed for its life cycle. Utilizing such a retroviraldelivery system for polynucleotides of the present invention will targetsaid gene and constructs to abnormally proliferating cells and willspare the non-dividing normal cells.

The polynucleotides of the present invention may be delivered directlyto cell proliferative disorder/disease sites in internal organs, bodycavities and the like by use of imaging devices used to guide aninjecting needle directly to the disease site. The polynucleotides ofthe present invention may also be administered to disease sites at thetime of surgical intervention.

By “cell proliferative disease” is meant any human or animal disease ordisorder, affecting any one or any combination of organs, cavities, orbody parts, which is characterized by single or multiple local abnormalproliferations of cells, groups of cells, or tissues, whether benign ormalignant.

Any amount of the polynucleotides of the present invention may beadministered as long as it has a biologically inhibiting effect on theproliferation of the treated cells. Moreover, it is possible toadminister more than one of the polynucleotide of the present inventionsimultaneously to the same site. By “biologically inhibiting” is meantpartial or total growth inhibition as well as decreases in the rate ofproliferation or growth of the cells. The biologically inhibitory dosemay be determined by assessing the effects of the polynucleotides of thepresent invention on target malignant or abnormally proliferating cellgrowth in tissue culture, tumor growth in animals and cell cultures, orany other method known to one of ordinary skill in the art.

The present invention is further directed to antibody-based therapieswhich involve administering of anti-polypeptides and anti-polynucleotideantibodies to a mammalian, preferably human, patient for treating,preventing, and/or diagnosing one or more of the described diseases,disorders, and/or conditions. Methods for producing anti-polypeptidesand anti-polynucleotide antibodies polyclonal and monoclonal antibodiesare described in detail elsewhere herein. Such antibodies may beprovided in pharmaceutically acceptable compositions as known in the artor as described herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

In particular, the antibodies, fragments and derivatives of the presentinvention are useful for treating, preventing, and/or diagnosing asubject having or developing cell proliferative and/or differentiationdiseases, disorders, and/or conditions as described herein. Suchtreatment comprises administering a single or multiple doses of theantibody, or a fragment, derivative, or a conjugate thereof.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors, for example, which serve toincrease the number or activity of effector cells which interact withthe antibodies.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of diseases, disorders, and/orconditions related to polynucleotides or polypeptides, includingfragments thereof, of the present invention. Such antibodies, fragments,or regions, will preferably have an affinity for polynucleotides orpolypeptides, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10-6M,10-6M, 5×10-7M, 10-7M, 5×10-8M, 10-8M, 5×10-9M, 10-9M, 5×10-10M, 10-10M,5×10-11M, 10-11M, 5×10-12M, 10-12M, 5×10-13M, 10-13M, 5×10-14M, 10-14M,5×10-15M, 10-15M.

Moreover, polypeptides of the present invention may be useful ininhibiting the angiogenesis of proliferative cells or tissues, eitheralone, as a protein fusion, or in combination with other polypeptidesdirectly or indirectly, as described elsewhere herein. In a mostpreferred embodiment, said anti-angiogenesis effect may be achievedindirectly, for example, through the inhibition of hematopoietic,tumor-specific cells, such as tumor-associated macrophages (See Joseph IB, et al. J Natl Cancer Inst, 90(21):1648-53 (1998), which is herebyincorporated by reference). Antibodies directed to polypeptides orpolynucleotides of the present invention may also result in inhibitionof angiogenesis directly, or indirectly (See Witte L, et al., CancerMetastasis Rev. 17(2):155-61 (1998), which is hereby incorporated byreference)).

Polypeptides, including protein fusions, of the present invention, orfragments thereof may be useful in inhibiting proliferative cells ortissues through the induction of apoptosis. Said polypeptides may acteither directly, or indirectly to induce apoptosis of proliferativecells and tissues, for example in the activation of a death-domainreceptor, such as tumor necrosis factor (TNF) receptor-1, CD95(Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) andTNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (SeeSchulze-Osthoff K, et al., Eur J Biochem 254(3):439-59 (1998), which ishereby incorporated by reference). Moreover, in another preferredembodiment of the present invention, said polypeptides may induceapoptosis through other mechanisms, such as in the activation of otherproteins which will activate apoptosis, or through stimulating theexpression of said proteins, either alone or in combination with smallmolecule drugs or adjuvants, such as apoptonin, galectins, thioredoxins,antiinflammatory proteins (See for example, Mutat. Res. 400(1-2):447-55(1998), Med Hypotheses.50(5):423-33 (1998), Chem. Biol. Interact Apr24;1 1-112:23-34 (1998), J Mol Med.76(6):402-12 (1998), Int. J. TissueReact. 20(1):3-15 (1998), which are all hereby incorporated byreference).

Polypeptides, including protein fusions to, or fragments thereof, of thepresent invention are useful in inhibiting the metastasis ofproliferative cells or tissues. Inhibition may occur as a direct resultof administering polypeptides, or antibodies directed to saidpolypeptides as described elsewhere herein, or indirectly, such asactivating the expression of proteins known to inhibit metastasis, forexample alpha 4 integrins, (See, e.g., Curr Top Microbiol Immunol1998;231:12541, which is hereby incorporated by reference). Suchtherapeutic affects of the present invention may be achieved eitheralone, or in combination with small molecule drugs or adjuvants.

In another embodiment, the invention provides a method of deliveringcompositions containing the polypeptides of the invention (e.g.,compositions containing polypeptides or polypeptide antibodiesassociated with heterologous polypeptides, heterologous nucleic acids,toxins, or prodrugs) to targeted cells expressing the polypeptide of thepresent invention. Polypeptides or polypeptide antibodies of theinvention may be associated with heterologous polypeptides, heterologousnucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionicand/or covalent interactions.

Polypeptides, protein fusions to, or fragments thereof, of the presentinvention are useful in enhancing the immunogenicity and/or antigenicityof proliferating cells or tissues, either directly, such as would occurif the polypeptides of the present invention ‘vaccinated’ the immuneresponse to respond to proliferative antigens and immunogens, orindirectly, such as in activating the expression of proteins known toenhance the immune response (e.g. chemokines), to said antigens andimmunogens.

Cardiovascular Disorders

Polynucleotides or polypeptides, or agonists or antagonists of theinvention may be used to treat, prevent, and/or diagnose cardiovasculardiseases, disorders, and/or conditions, including peripheral arterydisease, such as limb ischemia.

Cardiovascular diseases, disorders, and/or conditions includecardiovascular abnormalities, such as arterio-arterial fistula,arteriovenous fistula, cerebral arteriovenous malformations, congenitalheart defects, pulmonary atresia, and Scimitar Syndrome. Congenitalheart defects include aortic coarctation, cor triatriatum, coronaryvessel anomalies, crisscross heart, dextrocardia, patent ductusarteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic leftheart syndrome, levocardia, tetralogy of fallot, transposition of greatvessels, double outlet right ventricle, tricuspid atresia, persistenttruncus arteriosus, and heart septal defects, such as aortopulmonaryseptal defect, endocardial cushion defects, Lutembacher's Syndrome,trilogy of Fallot, ventricular heart septal defects.

Cardiovascular diseases, disorders, and/or conditions also include heartdisease, such as arrhythmias, carcinoid heart disease, high cardiacoutput, low cardiac output, cardiac tamponade, endocarditis (includingbacterial), heart aneurysm, cardiac arrest, congestive heart failure,congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, hearthypertrophy, congestive cardiomyopathy, left ventricular hypertrophy,right ventricular hypertrophy, post-infarction heart rupture,ventricular septal rupture, heart valve diseases, myocardial diseases,myocardial ischemia, pericardial effusion, pericarditis (includingconstrictive and tuberculous), pneumopericardium, postpericardiotomysyndrome, pulmonary heart disease, rheumatic heart disease, ventriculardysfunction, hyperemia, cardiovascular pregnancy complications, ScimitarSyndrome, cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias include sinus arrhythmia, atrial fibrillation, atrialflutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branchblock, sinoatrial block, long QT syndrome, parasystole,Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome,Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, andventricular fibrillation. Tachycardias include paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

Heart valve disease include aortic valve insufficiency, aortic valvestenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse,tricuspid valve prolapse, mitral valve insufficiency, mitral valvestenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonaryvalve stenosis, tricuspid atresia, tricuspid valve insufficiency, andtricuspid valve stenosis.

Myocardial diseases include alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardialfibrosis, Kearns Syndrome, myocardial reperfusion injury, andmyocarditis.

Myocardial ischemias include coronary disease, such as angina pectoris,coronary aneurysm, coronary arteriosclerosis, coronary thrombosis,coronary vasospasm, myocardial infarction and myocardial stunning.

Cardiovascular diseases also include vascular diseases such asaneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-WeberSyndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis,aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis,enarteritis, polyarteritis nodosa, cerebrovascular diseases, disorders,and/or conditions, diabetic angiopathies, diabetic retinopathy,embolisms, thrombosis, erythromelalgia, hemorrhoids, hepaticveno-occlusive disease, hypertension, hypotension, ischemia, peripheralvascular diseases, phlebitis, pulmonary veno-occlusive disease,Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitarsyndrome, superior vena cava syndrome, telangiectasia, ataciatelangiectasia, hereditary hemorrhagic telangiectasia, varicocele,varicose veins, varicose ulcer, vasculitis, and venous insufficiency.

Aneurysms include dissecting aneurysms, false aneurysms, infectedaneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms,coronary aneurysms, heart aneurysms, and iliac aneurysms.

Arterial occlusive diseases include arteriosclerosis, intermittentclaudication, carotid stenosis, fibromuscular dysplasias, mesentericvascular occlusion, Moyamoya disease, renal artery obstruction, retinalartery occlusion, and thromboangiitis obliterans.

Cerebrovascular diseases, disorders, and/or conditions include carotidartery diseases, cerebral amyloid angiopathy, cerebral aneurysm,cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenousmalformation, cerebral artery diseases, cerebral embolism andthrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg'ssyndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma,subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia(including transient), subclavian steal syndrome, periventricularleukomalacia, vascular headache, cluster headache, migraine, andvertebrobasilar insufficiency.

Embolisms include air embolisms, amniotic fluid embolisms, cholesterolembolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, andthromoboembolisms. Thrombosis include coronary thrombosis, hepatic veinthrombosis, retinal vein occlusion, carotid artery thrombosis, sinusthrombosis, Wallenberg's syndrome, and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, compartmentsyndromes, anterior compartment syndrome, myocardial ischemia,reperfusion injuries, and peripheral limb ischemia. Vasculitis includesaortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome,mucocutaneous lymph node syndrome, thromboangiitis obliterans,hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergiccutaneous vasculitis, and Wegener's granulomatosis.

Polynucleotides or polypeptides, or agonists or antagonists of theinvention, are especially effective for the treatment of critical limbischemia and coronary disease.

Polypeptides may be administered using any method known in the art,including, but not limited to, direct needle injection at the deliverysite, intravenous injection, topical administration, catheter infusion,biolistic injectors, particle accelerators, gelfoam sponge depots, othercommercially available depot materials, osmotic pumps, oral orsuppositorial solid pharmaceutical formulations, decanting or topicalapplications during surgery, aerosol delivery. Such methods are known inthe art. Polypeptides of the invention may be administered as part of aTherapeutic, described in more detail below. Methods of deliveringpolynucleotides of the invention are described in more detail herein.

Diseases at the Cellular Level

Diseases associated with increased cell survival or the inhibition ofapoptosis that could be treated, prevented, and/or diagnosed by thepolynucleotides or polypeptides and/or antagonists or agonists of theinvention, include cancers (such as follicular lymphomas, carcinomaswith p53 mutations, and hormone-dependent tumors, including, but notlimited to colon cancer, cardiac tumors, pancreatic cancer, melanoma,retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicularcancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma,endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune diseases, disorders, and/orconditions (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto'sthyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) and viral infections (suchas herpes viruses, pox viruses and adenoviruses), inflammation, graft v.host disease, acute graft rejection, and chronic graft rejection. Inpreferred embodiments, the polynucleotides or polypeptides, and/oragonists or antagonists of the invention are used to inhibit growth,progression, and/or metastasis of cancers, in particular those listedabove.

Additional diseases or conditions associated with increased cellsurvival that could be treated, prevented or diagnosed by thepolynucleotides or poly eptides, or agonists or antagonists of theinvention, include, but are not limited to, progression, and/ormetastases of malignancies and related disorders such as leukemia(including acute leukemias (e.g., acute lymphocytic leukemia, acutemyelocytic leukemia (including myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias(e.g., chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, and solid tumors including, butnot limited to, sarcomas and carcinomas such as fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

Diseases associated with increased apoptosis that could be treated,prevented, and/or diagnosed by the polynucleotides or polypeptides,and/or agonists or antagonists of the invention, include AIDS;neurodegenerative diseases, disorders, and/or conditions (such asAlzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis,Retinitis pigmentosa, Cerebellar degeneration and brain tumor or priorassociated disease); autoimmune diseases, disorders, and/or conditions(such as, multiple sclerosis, Sjogren's syndrome, Hashimoto'sthyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes(such as aplastic anemia), graft v. host disease, ischemic injury (suchas that caused by myocardial infarction, stroke and reperfusion injury),liver injury (e.g., hepatitis related liver injury, ischemia/reperfusioninjury, cholestosis (bile duct injury) and liver cancer);: toxin-inducedliver disease (such as that caused by alcohol), septic shock, cachexiaand anorexia.

Wound Healing and Epithelial Cell Proliferation

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing the polynucleotides or polypeptides,and/or agonists or antagonists of the invention, for therapeuticpurposes, for example, to stimulate epithelial cell proliferation andbasal keratinocytes for the purpose of wound healing, and to stimulatehair follicle production and healing of dermal wounds. Polynucleotidesor polypeptides, as well as agonists or antagonists of the invention,may be clinically useful in stimulating wound healing including surgicalwounds, excisional wounds, deep wounds involving damage of the dermisand epidermis, eye tissue wounds, dental tissue wounds, oral cavitywounds, diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers,venous stasis ulcers, burns resulting from heat exposure or chemicals,and other abnormal wound healing conditions such as uremia,malnutrition, vitamin deficiencies and complications associated withsystemic treatment with steroids, radiation therapy and antineoplasticdrugs and antimetabolites. Polynucleotides or polypeptides, and/oragonists or antagonists of the invention, could be used to promotedermal reestablishment subsequent to dermal loss

The polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, could be used to increase the adherence of skin grafts toa wound bed and to stimulate re-epithelialization from the wound bed.The following are a non-exhaustive list of grafts that polynucleotidesor polypeptides, agonists or antagonists of the invention, could be usedto increase adherence to a wound bed: autografts, artificial skin,allografts, autodermic graft, autoepidermic grafts, avacular grafts,Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft,delayed graft, dermic graft, epidermic graft, fascia graft, fullthickness graft, heterologous graft, xenograft, homologous graft,hyperplastic graft, lamellar graft, mesh graft, mucosal graft,Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft,penetrating graft, split skin graft, thick split graft. Thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, can be used to promote skin strength and to improve theappearance of aged skin.

It is believed that the polynucleotides or polypeptides, and/or agonistsor antagonists of the invention, will also produce changes in hepatocyteproliferation, and epithelial cell proliferation in the lung, breast,pancreas, stomach, small intestine, and large intestine. Thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could promote proliferation of epithelial cells such assebocytes, hair follicles, hepatocytes, type II pneumocytes,mucin-producing goblet cells, and other epithelial cells and theirprogenitors contained within the skin, lung, liver, and gastrointestinaltract. The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, may promote proliferation of endothelialcells, keratinocytes, and basal keratinocytes.

The polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, could also be used to reduce the side effects of guttoxicity that result from radiation, chemotherapy treatments or viralinfections. The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, may have a cytoprotective effect on thesmall intestine mucosa. The polynucleotides or polypeptides, and/oragonists or antagonists of the invention, may also stimulate healing ofmucositis (mouth ulcers) that result from chemotherapy and viralinfections.

The polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, could further be used in full regeneration of skin infull and partial thickness skin defects, including burns, (i.e.,repopulation of hair follicles, sweat glands, and sebaceous glands),treatment of other skin defects such as psoriasis. The polynucleotidesor polypeptides, and/or agonists or antagonists of the invention, couldbe used to treat epidermolysis bullosa, a defect in adherence of theepidermis to the underlying dermis which results in frequent, open andpainful blisters by accelerating reepithelialization of these lesions.The polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, could also be used to treat gastric and doudenal ulcersand help heal by scar formation of the mucosal lining and regenerationof glandular mucosa and duodenal mucosal lining more rapidly.Inflamamatory bowel diseases, such as Crohn's disease and ulcerativecolitis, are diseases which result in destruction of the mucosal surfaceof the small or large intestine, respectively. Thus, the polynucleotidesor polypeptides, and/or agonists or antagonists of the invention, couldbe used to promote the resurfacing of the mucosal surface to aid morerapid healing and to prevent progression of inflammatory bowel disease.Treatment with the polynucleotides or polypeptides, and/or agonists orantagonists of the invention, is expected to have a significant effecton the production of mucus throughout the gastrointestinal tract andcould be used to protect the intestinal mucosa from injurious substancesthat are ingested or following surgery. The polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, could beused to treat diseases associate with the under expression of thepolynucleotides of the invention.

Moreover, the polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could be used to prevent and heal damageto the lungs due to various pathological states. A growth factor such asthe polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, which could stimulate proliferation and differentiationand promote the repair of alveoli and brochiolar epithelium to preventor treat acute or chronic lung damage. For example, emphysema, whichresults in the progressive loss of aveoli, and inhalation injuries,i.e., resulting from smoke inhalation and burns, that cause necrosis ofthe bronchiolar epithelium and alveoli could be effectively treated,prevented, and/or diagnosed using the polynucleotides or polypeptides,and/or agonists or antagonists of the invention. Also, thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could be used to stimulate the proliferation of anddifferentiation of type II pneumocytes, which may help treat or preventdisease such as hyaline membrane diseases, such as infant respiratorydistress syndrome and bronchopulmonary displasia, in premature infants.

The polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, could stimulate the proliferation and differentiation ofhepatocytes and, thus, could be used to alleviate or treat liverdiseases and pathologies such as fulminant liver failure caused bycirrhosis, liver damage caused by viral hepatitis and toxic substances(i.e., acetaminophen, carbon tetraholoride and other hepatotoxins knownin the art).

In addition, the polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could be used treat or prevent the onsetof diabetes mellitus. In patients with newly diagnosed Types I and Hdiabetes, where some islet cell function remains, the polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, could beused to maintain the islet function so as to alleviate, delay or preventpermanent manifestation of the disease. Also, the polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, could beused as an auxiliary in islet cell transplantation to improve or promoteislet cell function.

Neurological Diseases

Nervous system diseases, disorders, and/or conditions, which can betreated, prevented, and/or diagnosed with the compositions of theinvention (e.g., polypeptides, polynucleotides, and/or agonists orantagonists), include, but are not limited to, nervous system injuries,and diseases, disorders, and/or conditions which result in either adisconnection of axons, a diminution or degeneration of neurons, ordemyelination. Nervous system lesions which may be treated, prevented,and/or diagnosed in a patient (including human and non-human mammalianpatients) according to the invention, include but are not limited to,the following lesions of either the central (including spinal cord,brain) or peripheral nervous systems: (1) ischemic lesions, in which alack of oxygen in a portion of the nervous system results in neuronalinjury or death, including cerebral infarction or ischemia, or spinalcord infarction or ischemia; (2) traumatic lesions, including lesionscaused by physical injury or associated with surgery, for example,lesions which sever a portion of the nervous system, or compressioninjuries; (3) malignant lesions, in which a portion of the nervoussystem is destroyed or injured by malignant tissue which is either anervous system associated malignancy or a malignancy derived fromnon-nervous system tissue; (4) infectious lesions, in which a portion ofthe nervous system is destroyed or injured as a result of infection, forexample, by an abscess or associated with infection by humanimmunodeficiency virus, herpes zoster, or herpes simplex virus or withLyme disease, tuberculosis, syphilis; (5) degenerative lesions, in whicha portion of the nervous system is destroyed or injured as a result of adegenerative process including but not limited to degenerationassociated with Parkinson's disease, Alzheimer's disease, Huntington'schorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associatedwith nutritional diseases, disorders, and/or conditions, in which aportion of the nervous system is destroyed or injured by a nutritionaldisorder or disorder of metabolism including but not limited to, vitaminB12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcoholamblyopia, Marchiafava-Bignami disease (primary degeneration of thecorpus callosum), and alcoholic cerebellar degeneration; (7)neurological lesions associated with systemic diseases including, butnot limited to, diabetes (diabetic neuropathy, Bell's palsy), systemiclupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused bytoxic substances including alcohol, lead, or particular neurotoxins; and(9) demyelinated lesions in which a portion of the nervous system isdestroyed or injured by a demyelinating disease including, but notlimited to, multiple sclerosis, human immunodeficiency virus-associatedmyelopathy, transverse myelopathy or various etiologies, progressivemultifocal leukoencephalopathy, and central pontine myelinolysis.

In a preferred embodiment, the polypeptides, polynucleotides, oragonists or antagonists of the invention are used to protect neuralcells from the damaging effects of cerebral hypoxia. According to thisembodiment, the compositions of the invention are used to treat,prevent, and/or diagnose neural cell injury associated with cerebralhypoxia. In one aspect of this embodiment, the polypeptides,polynucleotides, or agonists or antagonists of the invention are used totreat, prevent, and/or diagnose neural cell injury associated withcerebral ischemia. In another aspect of this embodiment, thepolypeptides, polynucleotides, or agonists or antagonists of theinvention are used to treat, prevent, and/or diagnose neural cell injuryassociated with cerebral infarction. In another aspect of thisembodiment, the polypeptides, polynucleotides, or agonists orantagonists of the invention are used to treat, prevent, and/or diagnoseor prevent neural cell injury associated with a stroke. In a furtheraspect of this embodiment, the polypeptides, polynucleotides, oragonists or antagonists of the invention are used to treat, prevent,and/or diagnose neural cell injury associated with a heart attack.

The compositions of the invention which are useful for treating orpreventing a nervous system disorder may be selected by testing forbiological activity in promoting the survival or differentiation ofneurons. For example, and not by way of limitation, compositions of theinvention which elicit any of the following effects may be usefulaccording to the invention: (1) increased survival time of neurons inculture; (2) increased sprouting of neurons in culture or in vivo; (3)increased production of a neuron-associated molecule in culture or invivo, e.g., choline acetyltransferase or acetylcholinesterase withrespect to motor neurons; or (4) decreased symptoms of neurondysfunction in vivo. Such effects may be measured by any method known inthe art. In preferred, non-limiting embodiments, increased survival ofneurons may routinely be measured using a method set forth herein orotherwise known in the art, such as, for example, the method set forthin Arakawa et al. (J. Neurosci. 10:3507-3515 (1990)); increasedsprouting of neurons may be detected by methods known in the art, suchas, for example, the methods set forth in Pestronk et al. (Exp. Neurol.70:65-82 (1980)) or Brown et al. (Ann. Rev. Neurosci. 4:1742 (1981));increased production of neuron-associated molecules may be measured bybioassay, enzymatic assay, antibody binding, Northern blot assay, etc.,using techniques known in the art and depending on the molecule to bemeasured; and motor neuron dysfunction may be measured by assessing thephysical manifestation of motor neuron disorder, e.g., weakness, motorneuron conduction velocity, or functional disability.

In specific embodiments, motor neuron diseases, disorders, and/orconditions that may be treated, prevented, and/or diagnosed according tothe invention include, but are not limited to, diseases, disorders,and/or conditions such as infarction, infection, exposure to toxin,trauma, surgical damage, degenerative disease or malignancy that mayaffect motor neurons as well as other components of the nervous system,as well as diseases, disorders, and/or conditions that selectivelyaffect neurons such as amyotrophic lateral sclerosis, and including, butnot limited to, progressive spinal muscular atrophy, progressive bulbarpalsy, primary lateral sclerosis, infantile and juvenile muscularatrophy, progressive bulbar paralysis of childhood (Fazio-Londesyndrome), poliomyelitis and the post polio syndrome, and HereditaryMotorsensory Neuropathy (Charcot-Marie-Tooth Disease).

Infectious Disease

A polypeptide or polynucleotide and/or agonist or antagonist of thepresent invention can be used to treat, prevent, and/or diagnoseinfectious agents. For example, by increasing the immune response,particularly increasing the proliferation and differentiation of Band/or T cells, infectious diseases may be treated, prevented, and/ordiagnosed. The immune response may be increased by either enhancing anexisting immune response, or by initiating a new immune response.Alternatively, polypeptide or polynucleotide and/or agonist orantagonist of the present invention may also directly inhibit theinfectious agent, without necessarily eliciting an immune response.

Viruses are one example of an infectious agent that can cause disease orsymptoms that can be treated, prevented, and/or diagnosed by apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention. Examples of viruses, include, but are not limited toExamples of viruses, include, but are not limited to the following DNAand RNA viruses and viral families: Arbovirus, Adenoviridae,Arenaviridae, Arterivinis, Bimaviridae, Bunyaviridae, Caliciviridae,Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae,Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus,Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae,Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A,Influenza B, and parainfluenza), Papiloma virus, Papovaviridae,Parvoviridae, Picomaviridae, Poxviridae (such as Smallpox or Vaccinia),Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II,Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling withinthese families can cause a variety of diseases or symptoms, including,but not limited to: arthritis, bronchiollitis, respiratory syncytialvirus, encephalitis, eye infections (e.g., conjunctivitis, keratitis),chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta),Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellowfever, meningitis, opportunistic infections (e.g., AIDS), pneumonia,Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps,Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella,sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts),and viremia. polynucleotides or polypeptides, or agonists or antagonistsof the invention, can be used to treat, prevent, and/or diagnose any ofthese symptoms or diseases. In specific embodiments, polynucleotides,polypeptides, or agonists or antagonists of the invention are used totreat, prevent, and/or diagnose: meningitis, Dengue, EBV, and/orhepatitis (e.g., hepatitis B). In an additional specific embodimentpolynucleotides, polypeptides, or agonists or antagonists of theinvention are used to treat patients nonresponsive to one or more othercommercially available hepatitis vaccines. In a further specificembodiment polynucleotides, polypeptides, or agonists or antagonists ofthe invention are used to treat, prevent, and/or diagnose AIDS.

Similarly, bacterial or fungal agents that can cause disease or symptomsand that can be treated, prevented, and/or diagnosed by a polynucleotideor polypeptide and/or agonist or antagonist of the present inventioninclude, but not limited to, include, but not limited to, the followingGram-Negative and Gram-positive bacteria and bacterial families andfungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium,Norcardia), Cryptococcus neoformans, Aspergillosis, Bacillaceae (e.g.,Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella,Borrelia (e.g., Borrelia burgdorferi), Brucellosis, Candidiasis,Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, E.coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli),Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, andSalmonella paratyphi), Serratia, Yersinia), Erysipelothrix,Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales,Mycobacterium leprae, Vibrio cholerae, Neisseriaceae (e.g.,Acinetobacter, Gonorrhea, Menigococcal), Meisseria meningitidis,Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g.,Heamophilus influenza type B), Pasteurella), Pseudomonas,Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp., Staphylococcal,Meningiococcal, Pneumococcal and Streptococcal (e.g., Streptococcuspneumoniae and Group B Streptococcus). These bacterial or fingalfamilies can cause the following diseases or symptoms, including, butnot limited to: bacteremia, endocarditis, eye infections(conjunctivitis, tuberculosis, uveitis), gingivitis, opportunisticinfections (e.g., AIDS related infections), paronychia,prosthesis-related infections, Reiter's Disease, respiratory tractinfections, such as Whooping Cough or Empyema, sepsis, Lyme Disease,Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning,Typhoid, pneumonia, Gonorrhea, meningitis (e.g., mengitis types A andB), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, RheumaticFever, Scarlet Fever, sexually transmitted diseases, skin diseases(e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections,wound infections. Polynucleotides or polypeptides, agonists orantagonists of the invention, can be used to treat, prevent, and/ordiagnose any of these symptoms or diseases. In specific embodiments,polynucleotides, polypeptides, agonists or antagonists of the inventionare used to treat, prevent, and/or diagnose: tetanus, Diptheria,botulism, and/or meningitis type B.

Moreover, parasitic agents causing disease or symptoms that can betreated, prevented, and/or diagnosed by a polynucleotide or polypeptideand/or agonist or antagonist of the present invention include, but notlimited to, the following families or class: Amebiasis, Babesiosis,Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic,Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis,Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax,Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale). Theseparasites can cause a variety of diseases or symptoms, including, butnot limited to: Scabies, Trombiculiasis, eye infections, intestinaldisease (e.g., dysentery, giardiasis), liver disease, lung disease,opportunistic infections (e.g., AIDS related), malaria, pregnancycomplications, and toxoplasmosis. polynucleotides or polypeptides, oragonists or antagonists of the invention, can be used totreat, prevent,and/or diagnose any of these symptoms or diseases. In specificembodiments, polynucleotides, polypeptides, or agonists or antagonistsof the invention are used to treat, prevent, and/or diagnose malaria.

Preferably, treatment or prevention using a polypeptide orpolynucleotide and/or agonist or antagonist of the present inventioncould either be by administering an effective amount of a polypeptide tothe patient, or by removing cells from the patient, supplying the cellswith a polynucleotide of the present invention, and returning theengineered cells to the patient (ex vivo therapy). Moreover, thepolypeptide or polynucleotide of the present invention can be used as anantigen in a vaccine to raise an immune response against infectiousdisease.

Regeneration

A polynucleotide or polypeptide and/or agonist or antagonist of thepresent invention can be used to differentiate, proliferate, and attractcells, leading to the regeneration of tissues. (See, Science 276:59-87(1997).) The regeneration of tissues could be used to repair, replace,or protect tissue damaged by congenital defects, trauma (wounds, burns,incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis,periodontal disease, liver failure), surgery, including cosmetic plasticsurgery, fibrosis, reperfusion injury, or systemic cytokine damage.

Tissues that could be regenerated using the present invention includeorgans (e.g., pancreas, liver, intestine, kidney, skin, endothelium),muscle (smooth, skeletal or cardiac), vasculature (including vascularand lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage,tendon, and ligament) tissue. Preferably, regeneration occurs without ordecreased scarring. Regeneration also may include angiogenesis.

Moreover, a polynucleotide or polypeptide and/or agonist or antagonistof the present invention may increase regeneration of tissues difficultto heal. For example, increased tendon/ligament regeneration wouldquicken recovery time after damage. A polynucleotide or polypeptideand/or agonist or antagonist of the present invention could also be usedprophylactically in an effort to avoid damage. Specific diseases thatcould be treated, prevented, and/or diagnosed include of tendinitis,carpal tunnel syndrome, and other tendon or ligament defects. A furtherexample of tissue regeneration of non-healing wounds includes pressureulcers, ulcers associated with vascular insufficiency, surgical, andtraumatic wounds.

Similarly, nerve and brain tissue could also be regenerated by using apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention to proliferate and differentiate nerve cells. Diseasesthat could be treated, prevented, and/or diagnosed using this methodinclude central and peripheral nervous system diseases, neuropathies, ormechanical and traumatic diseases, disorders, and/or conditions (e.g.,spinal cord disorders, head trauma, cerebrovascular disease, and stoke).Specifically, diseases associated with peripheral nerve injuries,peripheral neuropathy (e.g., resulting from chemotherapy or othermedical therapies), localized neuropathies, and central nervous systemdiseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington'sdisease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), couldall be treated, prevented, and/or diagnosed using the polynucleotide orpolypeptide and/or agonist or antagonist of the present invention.

Binding Activity

A polypeptide of the present invention may be used to screen formolecules that bind to the polypeptide or for molecules to which thepolypeptide binds. The binding of the polypeptide and the molecule mayactivate (agonist), increase, inhibit (antagonist), or decrease activityof the polypeptide or the molecule bound. Examples of such moleculesinclude antibodies, oligonucleotides, proteins (e.g., receptors),orsmall molecules.

Preferably, the molecule is closely related to the natural ligand of thepolypeptide, e.g., a fragment of the ligand, or a natural substrate, aligand, a structural or functional mimetic. (See, Coligan et al.,Current Protocols in Immunology 1(2):Chapter 5 (1991).) Similarly, themolecule can be closely related to the natural receptor to which thepolypeptide binds, or at least, a fragment of the receptor capable ofbeing bound by the polypeptide (e.g., active site). In either case, themolecule can be rationally designed using known techniques.

Preferably, the screening for these molecules involves producingappropriate cells which express the polypeptide, either as a secretedprotein or on the cell membrane. Preferred cells include cells frommammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide(or cell membrane containing the expressed polypeptide) are thenpreferably contacted with a test compound potentially containing themolecule to observe binding, stimulation, or inhibition of activity ofeither the polypeptide or the molecule.

The assay may simply test binding of a candidate compound to thepolypeptide, wherein binding is detected by a label, or in an assayinvolving competition with a labeled competitor. Further, the assay maytest whether the candidate compound results in a signal generated bybinding to the polypeptide.

Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining a polypeptide, measuring polypeptide/molecule activity orbinding, and comparing the polypeptide/molecule activity or binding to astandard.

Preferably, an ELISA assay can measure polypeptide level or activity ina sample (e.g., biological sample) using a monoclonal or polyclonalantibody. The antibody can measure polypeptide level or activity byeither binding, directly or indirectly, to the polypeptide or bycompeting with the polypeptide for a substrate.

Additionally, the receptor to which a polypeptide of the invention bindscan be identified by numerous methods known to those of skill in theart, for example, ligand panning and FACS sorting (Coligan, et al.,Current Protocols in lmuun., 1(2), Chapter 5, (1991)). For example,expression cloning is employed wherein polyadenylated RNA is preparedfrom a cell responsive to the polypeptides, for example, NIH3T3 cellswhich are known to contain multiple receptors for the FGF familyproteins, and SC-3 cells, and a cDNA library created from this RNA isdivided into pools and used to transfect COS cells or other cells thatare not responsive to the polypeptides. Transfected cells which aregrown on glass slides are exposed to the polypeptide of the presentinvention, after they have been labeled. The polypeptides can be labeledby a variety of means including iodination or inclusion of a recognitionsite for a site-specific protein kinase.

Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clones that encodesthe putative receptor.

As an alternative approach for receptor identification, the labeledpolypeptides can be photoaffinity linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE analysis and exposed to X-ray film. The labeledcomplex containing the receptors of the polypeptides can be excised,resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

Moreover, the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”) may be employed to modulate the activities of polypeptidesof the invention thereby effectively generating agonists and antagonistsof polypeptides of the invention. See generally, U.S. Pat. Nos.5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten,P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S.Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al., J. Mol.Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques24(2):308-13 (1998) (each of these patents and publications are herebyincorporated by reference). In one embodiment, alteration ofpolynucleotides and corresponding polypeptides of the invention may beachieved by DNA shuffling. DNA shuffling involves the assembly of two ormore DNA segments into a desired polynucleotide sequence of theinvention molecule by homologous, or site-specific, recombination. Inanother embodiment, polynucleotides and corresponding polypeptides ofthe invention may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of the polypeptides of theinvention may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules. In preferred embodiments, the heterologous molecules arefamily members. In further preferred embodiments, the heterologousmolecule is a growth factor such as, for example, platelet-derivedgrowth factor (PDGF), insulin-like growth factor (IGF-I), transforminggrowth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblastgrowth factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2, BMP4,BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A, OP-2,dorsalin, growth differentiation factors (GDFs), nodal, MIS,inhibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, andglial-derived neurotrophic factor (GDNF).

Other preferred fragments are biologically active fragments of thepolypeptides of the invention. Biologically active fragments are thoseexhibiting activity similar, but not necessarily identical, to anactivity of the polypeptide. The biological activity of the fragmentsmay include an improved desired activity, or a decreased undesirableactivity.

Additionally, this invention provides a method of screening compounds toidentify those which modulate the action of the polypeptide of thepresent invention. An example of such an assay comprises combining amammalian fibroblast cell, a the polypeptide of the present invention,the compound to be screened and 3[H] thymidine under cell cultureconditions where the fibroblast cell would normally proliferate. Acontrol assay may be performed in the absence of the compound to bescreened and compared to the amount of fibroblast proliferation in thepresence of the compound to determine if the compound stimulatesproliferation by determining the uptake of 3[H] thymidine in each case.The amount of fibroblast cell proliferation is measured by liquidscintillation chromatography which measures the incorporation of 3[H]thymidine. Both agonist and antagonist compounds may be identified bythis procedure.

In another method, a mammalian cell or membrane preparation expressing areceptor for a polypeptide of the present invention is incubated with alabeled polypeptide of the present invention in the presence of thecompound. The ability of the compound to enhance or block thisinteraction could then be measured. Alternatively, the response of aknown second messenger system following interaction of a compound to bescreened and the receptor is measured and the ability of the compound tobind to the receptor and elicit a second messenger response is measuredto determine if the compound is a potential agonist or antagonist. Suchsecond messenger systems include but are not limited to, cAMP guanylatecyclase, ion channels or phosphoinositide hydrolysis.

All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat, prevent, and/or diagnose disease or to bring about a particularresult in a patient (e.g., blood vessel growth) by activating orinhibiting the polypeptide/molecule. Moreover, the assays can discoveragents which may inhibit or enhance the production of the polypeptidesof the invention from suitably manipulated cells or tissues. Therefore,the invention includes a method of identifying compounds which bind tothe polypeptides of the invention comprising the steps of: (a)incubating a candidate binding compound with the polypeptide; and (b)determining if binding has occurred. Moreover, the invention includes amethod of identifying agonists/antagonists comprising the steps of: (a)incubating a candidate compound with the polypeptide, (b) assaying abiological activity, and (b) determining if a biological activity of thepolypeptide has been altered.

Also, one could identify molecules bind a polypeptide of the inventionexperimentally by using the beta-pleated sheet regions contained in thepolypeptide sequence of the protein. Accordingly, specific embodimentsof the invention are directed to polynucleotides encoding polypeptideswhich comprise, or alternatively consist of, the amino acid sequence ofeach beta pleated sheet regions in a disclosed polypeptide sequence.Additional embodiments of the invention are directed to polynucleotidesencoding polypeptides which comprise, or alternatively consist of, anycombination or all of contained in the polypeptide sequences of theinvention. Additional preferred embodiments of the invention aredirected to polypeptides which comprise, or alternatively consist of,the amino acid sequence of each of the beta pleated sheet regions in oneof the polypeptide sequences of the invention. Additional embodiments ofthe invention are directed to polypeptides which comprise, oralternatively consist of, any combination or all of the beta pleatedsheet regions in one of the polypeptide sequences of the invention.

Targeted Delivery

In another embodiment, the invention provides a method of deliveringcompositions to targeted cells expressing a receptor for a polypeptideof the invention, or cells expressing a cell bound form of a polypeptideof the invention.

As discussed herein, polypeptides or antibodies of the invention may beassociated with heterologous polypeptides, heterologous nucleic acids,toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalentinteractions. In one embodiment, the invention provides a method for thespecific delivery of compositions of the invention to cells byadministering polypeptides of the invention (including antibodies) thatare associated with heterologous polypeptides or nucleic acids. In oneexample, the invention provides a method for delivering a therapeuticprotein into the targeted cell. In another example, the inventionprovides a method for delivering a single stranded nucleic acid (e.g.,antisense or ribozymes) or double stranded nucleic acid (e.g., DNA thatcan integrate into the cell's genome or replicate episomally and thatcan be transcribed) into the targeted cell.

In another embodiment, the invention provides a method for the specificdestruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention (e.g., polypeptides of theinvention or antibodies of the invention) in association with toxins orcytotoxic prodrugs.

By “toxin” is meant compounds that bind and activate endogenouscytotoxic effector systems, radioisotopes, holotoxins, modified toxins,catalytic subunits of toxins, or any molecules or enzymes not normallypresent in or on the surface of a cell that under defined conditionscause the cell's death. Toxins that may be used according to the methodsof the invention include, but are not limited to, radioisotopes known inthe art, compounds such as, for example, antibodies (or complementfixing containing portions thereof) that bind an inherent or inducedendogenous cytotoxic effector system, thymidine kinase, endonuclease,RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheriatoxin, saporin, momordin, gelonin, pokeweed antiviral protein,alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant anon-toxic compound that is converted by an enzyme, normally present inthe cell, into a cytotoxic compound. Cytotoxic prodrugs that may be usedaccording to the methods of the invention include, but are not limitedto, glutamyl derivatives of benzoic acid mustard alkylating agent,phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside,daunorubisin, and phenoxyacetamide derivatives of doxorubicin.

Drug Screening

Further contemplated is the use of the polypeptides of the presentinvention, or the polynucleotides encoding these polypeptides, to screenfor molecules which modify the activities of the polypeptides of thepresent invention. Such a method would include contacting thepolypeptide of the present invention with a selected compound(s)suspected of having antagonist or agonist activity, and assaying theactivity of these polypeptides following binding.

This invention is particularly useful for screening therapeuticcompounds by using the polypeptides of the present invention, or bindingfragments thereof, in any of a variety of drug screening techniques. Thepolypeptide or fragment employed in such a test may be affixed to asolid support, expressed on a cell surface, free in solution, or locatedintracellularly. One method of drug screening utilizes eukaryotic orprokaryotic host cells which are stably transformed with recombinantnucleic acids expressing the polypeptide or fragment. Drugs are screenedagainst such transformed cells in competitive binding assays. One maymeasure, for example, the formulation of complexes between the agentbeing tested and a polypeptide of the present invention.

Thus, the present invention provides methods of screening for drugs orany other agents which affect activities mediated by the polypeptides ofthe present invention. These methods comprise contacting such an agentwith a polypeptide of the present invention or a fragment thereof andassaying for the presence of a complex between the agent and thepolypeptide or a fragment thereof, by methods well known in the art. Insuch a competitive binding assay, the agents to screen are typicallylabeled. Following incubation, free agent is separated from that presentin bound form, and the amount of free or uncomplexed label is a measureof the ability of a particular agent to bind to the polypeptides of thepresent invention.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to the polypeptides ofthe present invention, and is described in great detail in EuropeanPatent Application 84/03564, published on Sep. 13, 1984, which isincorporated herein by reference herein. Briefly stated, large numbersof different small peptide test compounds are synthesized on a solidsubstrate, such as plastic pins or some other surface. The peptide testcompounds are reacted with polypeptides of the present invention andwashed. Bound polypeptides are then detected by methods well known inthe art. Purified polypeptides are coated directly onto plates for usein the aforementioned drug screening techniques. In addition,non-neutralizing antibodies may be used to capture the peptide andimmobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding polypeptidesof the present invention specifically compete with a test compound forbinding to the polypeptides or fragments thereof. In this manner, theantibodies are used to detect the presence of any peptide which sharesone or more antigenic epitopes with a polypeptide of the invention.

The human CAN-12 polypeptides and/or peptides of the present invention,or immunogenic fragments or oligopeptides thereof, can be used forscreening therapeutic drugs or compounds in a variety of drug screeningtechniques. The fragment employed in such a screening assay may be freein solution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The reduction or abolition of activity of theformation of binding complexes between the ion channel protein and theagent being tested can be measured. Thus, the present invention providesa method for screening or assessing a plurality of compounds for theirspecific binding affinity with a CAN-12 polypeptide, or a bindablepeptide fragment, of this invention, comprising providing a plurality ofcompounds, combining the CAN-12 polypeptide, or a bindable peptidefragment, with each of a plurality of compounds for a time sufficient toallow binding under suitable conditions and detecting binding of theCAN-12 polypeptide or peptide to each of the plurality of testcompounds, thereby identifying the compounds that specifically bind tothe CAN-12 polypeptide or peptide.

Methods of identifying compounds that modulate the activity of the novelhuman CAN-12 polypeptides and/or peptides are provided by the presentinvention and comprise combining a potential or candidate compound ordrug modulator of calpain biological activity with an CAN-12 polypeptideor peptide, for example, the CAN-12 amino acid sequence as set forth inSEQ ID NOS:2, and measuring an effect of the candidate compound or drugmodulator on the biological activity of the CAN-12 polypeptide orpeptide. Such measurable effects include, for example, physical bindinginteraction; the ability to cleave a suitable calpain substrate; effectson native and cloned CAN-12-expressing cell line; and effects ofmodulators or other calpain-mediated physiological measures.

Another method of identifying compounds that modulate the biologicalactivity of the novel CAN-12 polypeptides of the present inventioncomprises combining a potential or candidate compound or drug modulatorof a calpain biological activity with a host cell that expresses theCAN-12 polypeptide and measuring an effect of the candidate compound ordrug modulator on the biological activity of the CAN-12 polypeptide. Thehost cell can also be capable of being induced to express the CAN-12polypeptide, e.g., via inducible expression. Physiological effects of agiven modulator candidate on the CAN-12 polypeptide can also bemeasured. Thus, cellular assays for particular calpain modulators may beeither direct measurement or quantification of the physical biologicalactivity of the CAN-1-2 polypeptide, or they may be measurement orquantification of a physiological effect. Such methods preferably employa CAN-12 polypeptide as described herein, or an overexpressedrecombinant CAN-12 polypeptide in suitable host cells containing anexpression vector as described herein, wherein the CAN-12 polypeptide isexpressed, overexpressed, or undergoes upregulated expression.

Another aspect of the present invention embraces a method of screeningfor a compound that is capable of modulating the biological activity ofa CAN-12 polypeptide, comprising providing a host cell containing anexpression vector harboring a nucleic acid sequence encoding a CAN-12polypeptide, or a functional peptide or portion thereof (e.g., SEQ IDNOS:2); determining the biological activity of the expressed CAN-12polypeptide in the absence of a modulator compound; contacting the cellwith the modulator compound and determining the biological activity ofthe expressed CAN-12 polypeptide in the presence of the modulatorcompound. In such a method, a difference between the activity of theCAN-12 polypeptide in the presence of the modulator compound and in theabsence of the modulator compound indicates a modulating effect of thecompound.

Essentially any chemical compound can be employed as a potentialmodulator or ligand in the assays according to the present invention.Compounds tested as calpain modulators can be any small chemicalcompound, or biological entity (e.g., protein, sugar, nucleic acid,lipid). Test compounds will typically be small chemical molecules andpeptides. Generally, the compounds used as potential modulators can bedissolved in aqueous or organic (e.g., DMSO-based) solutions. The assaysare designed to screen large chemical libraries by automating the assaysteps and providing compounds from any convenient source. Assays aretypically run in parallel, for example, in microtiter formats onmicrotiter plates in robotic assays. There are many suppliers ofchemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St.Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-BiochemicaAnalytika (Buchs, Switzerland), for example. Also, compounds may besynthesized by methods known in the art.

High throughput screening methodologies are particularly envisioned forthe detection of modulators of the novel CAN-12 polynucleotides andpolypeptides described herein. Such high throughput screening methodstypically involve providing a combinatorial chemical or peptide librarycontaining a large number of potential therapeutic compounds (e.g.,ligand or modulator compounds). Such combinatorial chemical libraries orligand libraries are then screened in one or more assays to identifythose library members (e.g., particular chemical species or subclasses)that display a desired characteristic activity. The compounds soidentified can serve as conventional lead compounds, or can themselvesbe used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated either by chemical synthesis or biologicalsynthesis, by combining a number of chemical building blocks (i.e.,reagents such as amino acids). As an example, a linear combinatoriallibrary, e.g., a polypeptide or peptide library, is formed by combininga set of chemical building blocks in every possible way for a givencompound length (i.e., the number of amino acids in a polypeptide orpeptide compound). Millions of chemical compounds can be synthesizedthrough such combinatorial mixing of chemical building blocks.

The preparation and screening of combinatorial chemical libraries iswell known to those having skill in the pertinent art. Combinatoriallibraries include, without limitation, peptide libraries (e.g. U.S. Pat.No. 5,010,175; Furka, 1991, Int. J. Pept. Prot. Res., 37:487-493; andHoughton et al., 1991, Nature, 354:84-88). Other chemistries forgenerating chemical diversity libraries can also be used. Nonlimitingexamples of chemical diversity library chemistries include, peptides(PCT Publication No. WO 91/019735), encoded peptides (PCT PublicationNo. WO 93/20242), random bio-oligomers (PCT Publication No. WO92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers suchas hydantoins, benzodiazepines and dipeptides (Hobbs et al., 1993, Proc.Natl. Acad. Sci. USA, 90:6909-6913), vinylogous polypeptides (Hagiharaet al., 1992, J. Amer. Chem. Soc., 114:6568), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., 1992, J.Amer. Chem. Soc., 114:9217-9218), analogous organic synthesis of smallcompound libraries (Chen et al., 1994, J. Amer. Chem. Soc., 116:2661),oligocarbamates (Cho et al., 1993, Science, 261:1303), and/or peptidylphosphonates (Campbell et al., 1994, J. Org. Chem., 59:658), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (U.S. Pat. No. 5,539,083), antibody libraries(e.g., Vaughn et al., 1996, Nature Biotechnology, 14(3):309-314) andPCT/US96/10287), carbohydrate libraries (e.g., Liang et al., 1996,Science, 274-1520-1522) and U.S. Pat. No. 5,593,853), small organicmolecule libraries (e.g., benzodiazepines, Baum C&EN, Jan. 18, 1993,page 33; and U.S. Pat. No. 5,288,514; isoprenoids, U.S. Pat. No.5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky.;Symphony, Rainin, Woburn, Mass.; 433A Applied Biosystems, Foster City,Calif.; 9050 Plus, Millipore, Bedford, Mass.). In addition, a largenumber of combinatorial libraries are commercially available (e.g.,ComGenex, Princeton, N.J.; Asinex, Moscow, Russia; Tripos, Inc., St.Louis, Mo.; ChemStar, Ltd., Moscow, Russia; 3D Pharmaceuticals, Exton,Pa.; Martek Biosciences, Columbia, Md., and the like).

In one embodiment, the invention provides solid phase based in vitroassays in a high throughput format, where the cell or tissue expressingan ion channel is attached to a solid phase substrate. In such highthroughput assays, it is possible to screen up to several thousanddifferent modulators or ligands in a single day. In particular, eachwell of a microtiter plate can be used to perform a separate assayagainst a selected potential modulator, or, if concentration orincubation time effects are to be observed, every 5-10 wells can test asingle modulator. Thus, a single standard microtiter plate can assayabout 96 modulators. If 1536 well plates are used, then a single platecan easily assay from about 100 to about 1500 different compounds. It ispossible to assay several different plates per day; thus, for example,assay screens for up to about 6,000-20,000 different compounds arepossible using the described integrated systems.

In another of its aspects, the present invention encompasses screeningand small molecule (e.g., drug) detection assays which involve thedetection or identification of small molecules that can bind to a givenprotein, i.e., a CAN-12 polypeptide or peptide. Particularly preferredare assays suitable for high throughput screening methodologies.

In such binding-based detection, identification, or screening assays, afunctional assay is not typically required. All that is needed is atarget protein, preferably substantially purified, and a library orpanel of compounds (e.g., ligands, drugs, small molecules) or biologicalentities to be screened or assayed for binding to the protein target.Preferably, most small molecules that bind to the target protein willmodulate activity in some manner, due to preferential, higher affinitybinding to functional areas or sites on the protein.

An example of such an assay is the fluorescence based thermal shiftassay (3-Dimensional Pharmaceuticals, Inc., 3DP, Exton, Pa.) asdescribed in U.S. Pat. Nos. 6,020,141 and 6,036,920 to Pantoliano etal.; see also, J. Zimmerman, 2000, Gen. Eng. News, 20(8)). The assayallows the detection of small molecules (e.g., drugs, ligands) that bindto expressed, and preferably purified, ion channel polypeptide based onaffinity of binding determinations by analyzing thermal unfolding curvesof protein-drug or ligand complexes. The drugs or binding moleculesdetermined by this technique can be further assayed, if desired, bymethods, such as those described herein, to determine if the moleculesaffect or modulate function or activity of the target protein.

To purify a CAN-12 polypeptide or peptide to measure a. biologicalbinding or ligand binding activity, the source may be a whole celllysate that can be prepared by successive freeze-thaw cycles (e.g., oneto three) in the presence of standard protease inhibitors. The CAN-12polypeptide may be partially or completely purified by standard proteinpurification methods, e.g., affinity chromatography using specificantibody described infra, or by ligands specific for an epitope tagengineered into the recombinant CAN-12 polypeptide molecule, also asdescribed herein. Binding activity can then be measured as described.

Compounds which are identified according to the methods provided herein,and which modulate or regulate the biological activity or physiology ofthe CAN-12 polypeptides according to the present invention are apreferred embodiment of this invention. It is contemplated that suchmodulatory compounds may be employed in treatment and therapeuticmethods for treating a condition that is mediated by the novel CAN-12polypeptides by administering to an individual in need of such treatmenta therapeutically effective amount of the compound identified by themethods described herein.

In addition, the present invention provides methods for treating anindividual in need of such treatment for a disease, disorder, orcondition that is mediated by the CAN-12 polypeptides of the invention,comprising administering to the individual a therapeutically effectiveamount of the CAN-12-modulating compound identified by a method providedherein.

Antisense And Ribozyme (Antagonists)

In specific embodiments, antagonists according to the present inventionare nucleic acids corresponding to the sequences contained in SEQ ID NO:1, 23, 53, and/or 55, or the complementary strand thereof, and/or tonucleotide sequences contained a deposited clone. In one embodiment,antisense sequence is generated internally by the organism, in anotherembodiment, the antisense sequence is separately administered (see, forexample, O'Connor, Neurochem., 56:560 (1991). Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988). Antisense technology can be used to control gene expressionthrough antisense DNA or RNA, or through triple-helix formation.Antisense techniques are discussed for example, in Okano, Neurochem.,56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance, Lee et al., Nucleic Acids Research,6:3073 (1979); Cooney et al., Science, 241:456 (1988); and Dervan etal., Science, 251:1300 (1991). The methods are based on binding of apolynucleotide to a complementary DNA or RNA.

For example, the use of c-myc and c-myb antisense RNA constructs toinhibit the growth of the non-lymphocytic leukemia cell line HL-60 andother cell lines was previously described. (Wickstrom et al. (1988);Anfossi et al. (1989)). These experiments were performed in vitro byincubating cells with the oligoribonucleotide. A similar procedure forin vivo use is described in WO 91/15580. Briefly, a pair ofoligonucleotides for a given antisense RNA is produced as follows: Asequence complimentary to the first 15 bases of the open reading frameis flanked by an EcoRl site on the 5 end and a HindIII site on the 3end. Next, the pair of oligonucleotides is heated at 90° C. for oneminute and then annealed in 2× ligation buffer (20 mM TRIS HCl pH 7.5,10 mM MgCl2, 100 MM dithiotlireitol (DTT) and 0.2 mM ATP) and thenligated to the EcoRl/Hind Im site of the retroviral vector PMV7 (WO91/15580).

For example, the 5′ coding portion of a polynucleotide that encodes themature polypeptide of the present invention may be used to design anantisense RNA oligonucleotide of from about 10 to 40 base pairs inlength. A DNA oligonucleotide is designed to be complementary to aregion of the gene involved in transcription thereby preventingtranscription and the production of the receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide.

In one embodiment, the antisense nucleic acid of the invention isproduced intracellularly by transcription from an exogenous sequence.For example, a vector or a portion thereof, is transcribed, producing anantisense nucleic acid (RNA) of the invention. Such a vector wouldcontain a sequence encoding the antisense nucleic acid of the invention.Such a vector can remain episomal or become chromosomally integrated, aslong as it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others known inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding a polypeptide of the invention, orfragments thereof, can be by any promoter known in the art to act invertebrate, preferably human cells. Such promoters can be inducible orconstitutive. Such promoters include, but are not limited to, the SV40early promoter region (Bernoist and Chambon, Nature, 29:304-310 (1981),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., Cell, 22:787-797 (1980), the herpes thymidinepromoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A., 78:1441-1445(1981), the regulatory sequences of the metallothionein gene (Brinsteret al., Nature, 296:39-42 (1982)), etc.

The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a gene ofinterest. However, absolute complementarity, although preferred, is notrequired. A sequence “complementary to at least a portion of an RNA,”referred to herein, means a sequence having sufficient complementarityto be able to hybridize with the RNA, forming a stable duplex; in thecase of double stranded antisense nucleic acids of the invention, asingle strand of the duplex DNA may thus be tested, or triplex formationmay be assayed. The ability to hybridize will depend on both the degreeof complementarity and the length of the antisense nucleic acidGenerally, the larger the hybridizing nucleic acid, the more basemismatches with a RNA sequence of the invention it may contain and stillform a stable duplex (or triplex as the case may be). One skilled in theart can ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message,e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., Nature,372:333-335 (1994). Thus, oligonucleotides complementary to either the5′- or 3′-non-translated, non-coding regions of a polynucleotidesequence of the invention could be used in an antisense approach toinhibit translation of endogenous mRNA. Oligonucleotides complementaryto the 5′ untranslated region of the mRNA should include the complementof the AUG start codon. Antisense oligonucleotides complementary to mRNAcoding regions are less efficient inhibitors of translation but could beused in accordance with the invention. Whether designed to hybridize tothe 5′-, 3′- or coding region of mRNA, antisense nucleic acids should beat least six nucleotides in length, and are preferably oligonucleotidesranging from 6 to about 50 nucleotides in length. In specific aspectsthe oligonucleotide is at least 10 nucleotides, at least 17 nucleotides,at least 25 nucleotides or at least 50 nucleotides.

The polynucleotides of the invention can be DNA or RNA or chimericmixtures or derivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. U.S.A.86:6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci., 84:648-652(1987); PCT Publication NO: WO88/09810, published Dec. 15, 1988) or theblood-brain barrier (see, e.g., PCT Publication NO: WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., BioTechniques, 6:958-976 (1988)) or intercalatingagents. (See, e.g., Zon, Pharm. Res., 5:539-549 (1988)). To this end,the oligonucleotide may be conjugated to another molecule, e.g., apeptide, hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacefic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group including,but not limited to, a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is ana-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual b-units, the strands run parallel to each other (Gautier et al.,Nucl. Acids Res., 15:6625-6641 (1987)). The oligonucleotide is a2-0-methylribonucleotide (Inoue et al., Nucl. Acids Res., 15:6131-6148(1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett.215:327-330 (1987)).

Polynucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (Nucl. Acids Res., 16:3209 (1988)),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci.U.S.A., 85:7448-7451 (1988)), etc.

While antisense nucleotides complementary to the coding region sequenceof the invention could be used, those complementary to the transcribeduntranslated region are most preferred.

Potential antagonists according to the invention also include catalyticRNA, or a ribozyme (See, e.g., PCT International Publication WO90/11364, published Oct. 4, 1990; Sarver et al, Scienice, 247:1222-1225(1990). While ribozymes that cleave mRNA at site specific recognitionsequences can be used to destroy muRNAs corresponding to thepolynucleotides of the invention, the use of hammerhead ribozymes ispreferred. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The sole requirement is that the target mRNA have the followingsequence of two bases: 5′-UG-3′. The construction and production ofhammerhead ribozymes is well known in the art and is described morefully in Haseloff and Gerlach, Nature, 334:585-591 (1988). There arenumerous potential hammerhead ribozyme cleavage sites within eachnucleotide sequence disclosed in the sequence listing. Preferably, theribozyme is engineered so that the cleavage recognition site is locatednear the 5′ end of the mRNA corresponding to the polynucleotides of theinvention; i.e., to increase efficiency and minimize the intracellularaccumulation of non-functional mRNA transcripts.

As in the antisense approach, the ribozymes of the invention can becomposed of modified oligonucleotides (e.g. for improved stability,targeting, etc.) and should be delivered to cells which express thepolynucleotides of the invention in vivo. DNA constructs encoding theribozyme may be introduced into the cell in the same manner as describedabove for the introduction of antisense encoding DNA. A preferred methodof delivery involves using a DNA construct “encoding” the ribozyme underthe control of a strong constitutive promoter, such as, for example, polIII or pol II promoter, so that transfected cells will producesufficient quantities of the ribozyme to destroy endogenous messages andinhibit translation. Since ribozymes unlike antisense molecules, arecatalytic, a lower intracellular concentration is required forefficiency.

Antagonist/agonist compounds may be employed to inhibit the cell growthand proliferation effects of the polypeptides of the present inventionon neoplastic cells and tissues, i.e. stimulation of angiogenesis oftumors, and, therefore, retard or prevent abnormal cellular growth andproliferation, for example, in tumor formation or growth.

The antagonist/agonist may also be employed to prevent hyper-vasculardiseases, and prevent the proliferation of epithelial lens cells afterextracapsular cataract surgery. Prevention of the mitogenic activity ofthe polypeptides of the present invention may also be desirous in casessuch as restenosis after balloon angioplasty.

The antagonist/agonist may also be employed to prevent the growth ofscar tissue during wound healing.

The antagonist/agonist may also be employed to treat, prevent, and/ordiagnose the diseases described herein.

Thus, the invention provides a method of treating or preventingdiseases, disorders, and/or conditions, including but not limited to thediseases, disorders, and/or conditions listed throughout thisapplication, associated with overexpression of a polynucleotide of thepresent invention by administering to a patient (a) an antisensemolecule directed to the polynucleotide of the present invention, and/or(b) a ribozyme directed to the polynucleotide of the present invention.

invention, and/or (b) a ribozyme directed to the polynucleotide of thepresent invention.

Biotic Associations

A polynucleotide or polypeptide and/or agonist or antagonist of thepresent invention may increase the organisms ability, either directly orindirectly, to initiate and/or maintain biotic associations with otherorganisms. Such associations may be symbiotic, nonsymbiotic,endosymbiotic, macrosymbiotic, and/or microsymbiotic in nature. Ingeneral, a polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention may increase the organisms ability to form bioticassociations with any member of the fungal, bacterial, lichen,mycorrhizal, cyanobacterial, dinoflaggellate, and/or algal, kingdom,phylums, families, classes, genuses, and/or species.

The mechanism by which a polynucleotide or polypeptide and/or agonist orantagonist of the present invention may increase the host organismsability, either directly or indirectly, to initiate and/or maintainbiotic associations is variable, though may include, modulatingosmolarity to desirable levels for the symbiont, modulating pH todesirable levels for the symbiont, modulating secretions of organicacids, modulating the secretion of specific proteins, phenoliccompounds, nutrients, or the increased expression of a protein requiredfor host-biotic organisms interactions (e.g., a receptor, ligand, etc.).Additional mechanisms are known in the art and are encompassed by theinvention (see, for example, “Microbial Signalling and Communication”,eds., R. England, G. Hobbs, N. Bainton, and D. McL. Roberts, CambridgeUniversity Press, Cambridge, (1999); which is hereby incorporated hereinby reference).

In an alternative embodiment, a polynucleotide or polypeptide and/oragonist or antagonist of the present invention may decrease the hostorganisms ability to form biotic associations with another organism,either directly or indirectly. The mechanism by which a polynucleotideor polypeptide and/or agonist or antagonist of the present invention maydecrease the host organisms ability, either directly or indirectly, toinitiate and/or maintain biotic associations with another organism isvariable, though may include, modulating osmolarity to undesirablelevels, modulating pH to undesirable levels, modulating secretions oforganic acids, modulating the secretion of specific proteins, phenoliccompounds, nutrients, or the decreased expression of a protein requiredfor host-biotic organisms interactions (e.g., a receptor, ligand, etc.).Additional mechanisms are known in the art and are encompassed by theinvention (see, for example, “Microbial Signalling and Communication”,eds., R. England, G. Hobbs, N. Bainton, and D. McL. Roberts, CambridgeUniversity Press, Cambridge, (1999); which is hereby incorporated hereinby reference).

The hosts ability to maintain biotic associations with a particularpathogen has significant implications for the overall health and fitnessof the host. For example, human hosts have symbiosis with entericbacteria in their gastrointestinal tracts, particularly in the small andlarge intestine. In fact, bacteria counts in feces of the distal colonoften approach 1012 per milliliter of feces. Examples of bowel flora inthe gastrointestinal tract are members of the Enterobacteriaceae,Bacteriodes, in addition to a-hemolytic streptococci, E. coli,Bifobacteria, Anaerobic cocci, Eubacteria, Costridia, lactobacilli, andyeasts. Such bacteria, among other things, assist the host in theassimilation of nutrients by breaking down food stuffs not typicallybroken down by the hosts digestive system, particularly in the hostsbowel. Therefore, increasing the hosts ability to maintain such a bioticassociation would help assure proper nutrition for the host.

Aberrations in the enteric bacterial population of mammals, particularlyhumans, has been associated with the following disorders: diarrhea,ileus, chronic inflammatory disease, bowel obstruction, duodenaldiverticula, biliary calculous disease, and malnutrition. Apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention are useful for treating, detecting, diagnosing,prognosing, and/or ameliorating, either directly or indirectly, and ofthe above mentioned diseases and/or disorders associated with aberrantenteric flora population.

The composition of the intestinal flora, for example, is based upon avariety of factors, which include, but are not limited to, the age,race, diet, malnutrition, gastric acidity, bile salt excretion, gutmotility, and immune mechanisms. As a result, the polynucleotides andpolypeptides, including agonists, antagonists, and fragments thereof,may modulate the ability of a host to form biotic associations byaffecting, directly or indirectly, at least one or more of thesefactors.

Although the predominate intestinal flora comprises anaerobic organisms,an underlying percentage represents aerobes (e.g., E. coli). This issignificant as such aerobes rapidly become the predominate organisms inintraabdominal infections—effectively becoming opportunistic early ininfection pathogenesis. As a result, there is an intrinsic need tocontrol aerobe populations, particularly for immune compromisedindividuals.

In a preferred embodiment, a polynucleotides and polypeptides, includingagonists, antagonists, and fragments thereof, are useful for inhibitingbiotic associations with specific enteric symbiont organisms in aneffort to control the population of such organisms.

Biotic associations occur not only in the gastrointestinal tract, butalso on an in the integument. As opposed to the gastrointestinal flora,the cutaneous flora is comprised almost equally with aerobic andanaerobic organisms. Examples of cutaneous flora are members of thegram-positive cocci (e.g., S. aureus, coagulase-negative staphylococci,micrococcus, M. sedentarius), gram-positive bacilli (e.g.,Corynebacterium species, C. minutissimum, Brevibacterium species,Propoionibacterium species, P. acnes), gram-negative bacilli (e.g.,Acinebacter species), and fuingi (Pityrosporum orbiculare). Therelatively low number of flora associated with the integument is basedupon the inability of many organisms to adhere to the skin. Theorganisms referenced above have acquired this unique ability. Therefore,the polynucleotides and polypeptides of the present invention may haveuses which include modulating the population of the cutaneous flora,either directly or indirectly.

Aberrations in the cutaneous flora are associated with a number ofsignificant diseases and/or disorders, which include, but are notlimited to the following: impetigo, ecthyma, blistering distaldactulitis, pustules, folliculitis, cutaneous abscesses, pittedkeratolysis, trichomycosis axcillaris, dermatophytosis complex, axillaryodor, erthyrasma, cheesy foot odor, acne, tinea versicolor, seborrheicdermititis, and Pityrosporum folliculitis, to name a few. Apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention are useful for treating, detecting, diagnosing,prognosing, and/or ameliorating, either directly or indirectly, and ofthe above mentioned diseases and/or disorders associated with aberrantcutaneous flora population.

Additional biotic associations, including diseases and disordersassociated with the aberrant growth of such associations, are known inthe art and are encompassed by the invention. See, for example,“Infectious Disease”, Second Edition, Eds., S. L., Gorbach, J. G.,Bartlett, and N. R., Blacklow, W. B. Saunders Company, Philadelphia,(1998); which is hereby incorporated herein by reference).

Pheromones

In another embodiment, a polynucleotide or polypeptide and/or agonist orantagonist of the present invention may increase the organisms abilityto synthesize and/or release a pheromone. Such a pheromone may, forexample, alter the organisms behavior and/or metabolism.

A polynucleotide or polypeptide and/or agonist or antagonist of thepresent invention may modulate the biosynthesis and/or release ofpheromones, the organisms ability to respond to pheromones (e.g.,behaviorally, and/or metabolically), and/or the organisms ability todetect pheromones. Preferably, any of the pheromones, and/or volatilesreleased from the organism, or induced, by a polynucleotide orpolypeptide and/or agonist or antagonist of the invention havebehavioral effects the organism.

Other Activities

The polypeptide of the present invention, as a result of the ability tostimulate vascular endothelial cell growth, may be employed in treatmentfor stimulating re-vascularization of ischemic tissues due to variousdisease conditions such as thrombosis, arteriosclerosis, and othercardiovascular conditions. These polypeptide may also be employed tostimulate angiogenesis and limb regeneration, as discussed above.

The polypeptide may also be employed for treating wounds due toinjuries, burns, post-operative tissue repair, and ulcers since they aremitogenic to various cells of different origins, such as fibroblastcells and skeletal muscle cells, and therefore, facilitate the repair orreplacement of damaged or diseased tissue.

The polypeptide of the present invention may also be employed stimulateneuronal growth and to treat, prevent, and/or diagnose neuronal damagewhich occurs in certain neuronal disorders or neuro-degenerativeconditions such as Alzheimer's disease, Parkinson's disease, andAIDS-related complex. The polypeptide of the invention may have theability to stimulate chondrocyte growth, therefore, they may be employedto enhance bone and periodontal regeneration and aid in tissuetransplants or bone grafts.

The polypeptide of the present invention may be also be employed toprevent skin aging due to sunburn by stimulating keratinocyte growth.

The polypeptide of the invention may also be employed for preventinghair loss, since FGF family members activate hair-forming cells andpromotes melano-cyte growth. Along the same lines, the polypeptides ofthe present invention may be employed to stimulate growth anddifferentiation of hematopoietic cells and bone marrow cells when usedin combination with other cytokines.

The polypeptide of the invention may also be employed to maintain organsbefore transplantation or for supporting cell culture of primarytissues.

The polypeptide of the present invention may also be employed forinducing tissue of mesodermal origin to differentiate in early embryos.

The polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may also increase or decrease the differentiation orproliferation of embryonic stem cells, besides, as discussed above,hematopoietic lineage.

The polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may also be used to modulate mammaliancharacteristics, such as body height, weight, hair color, eye color,skin, percentage of adipose tissue, pigmentation, size, and shape (e.g.,cosmetic surgery). Similarly, polypeptides or polynucleotides and/oragonist or antagonists of the present invention may be used to modulatemammalian metabolism affecting catabolism, anabolism, processing,utilization, and storage of energy.

Polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may be used to change a mammal's mental state orphysical state by influencing biorhythms, caricadic rhythms, depression(including depressive diseases, disorders, and/or conditions), tendencyfor violence, tolerance for pain, reproductive capabilities (preferablyby Activin or Inhibin-like activity), hormonal or endocrine levels,appetite, libido, memory, stress, or other cognitive qualities.

Polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may also be used to increase the efficacy of apharmaceutical composition, either directly or indirectly. Such a usemay be administered in simultaneous conjunction with saidpharmaceutical, or separately through either the same or different routeof administration (e.g., intravenous for the polynucleotide orpolypeptide of the present invention, and orally for the pharmaceutical,among others described herein.).

Polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may also be used to prepare individuals forextraterrestrial travel, low gravity environments, prolonged exposure toextraterrestrial radiation levels, low oxygen levels, reduction ofmetabolic activity, exposure to extraterrestrial pathogens, etc. Such ause may be administered either prior to an extraterrestrial event,during an extraterrestrial event, or both. Moreover, such a use mayresult in a number of beneficial changes in the recipient, such as, forexample, any one of the following, non-limiting, effects: an increasedlevel of hematopoietic cells, particularly red blood cells which wouldaid the recipient in coping with low oxygen levels; an increased levelof B-cells, T-cells, antigen presenting cells, and/or macrophages, whichwould aid the recipient in coping with exposure to extraterrestrialpathogens, for example; a temporary (i.e., reversible) inhibition ofhematopoietic cell production which would aid the recipient in copingwith exposure to extraterrestrial radiation levels; increase and/orstability of bone mass which would aid the recipient in coping with lowgravity environments; and/or decreased metabolism which wouldeffectively facilitate the recipients ability to prolong theirextraterrestrial travel by any one of the following, non-limiting means:(i) aid the recipient by decreasing their basal daily energyrequirements; (ii) effectively lower the level of oxidative and/ormetabolic stress in recipient (i.e., to enable recipient to cope withincreased extraterrestial radiation levels by decreasing the level ofinternal oxidative/metabolic damage acquired during normal basal energyrequirements; and/or (iii) enabling recipient to subsist at a lowermetabolic temperature (i.e., cryogenic, and/or sub-cryogenicenvironment).

Polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may also be used as a food additive or preservative,such as to increase or decrease storage capabilities, fat content,lipid, protein, carbohydrate, vitamins, minerals, cofactors or othernutritional components.

Other Preferred Embodiments

Further preferred is a method of making a recombinant vector comprisinginserting any of the above isolated nucleic acid molecule(s) into avector. Also preferred is the recombinant vector produced by thismethod. Also preferred is a method of making a recombinant host cellcomprising introducing the vector into a host cell, as well as therecombinant host cell produced by this method.

Also preferred is a method of making an isolated polypeptide comprisingculturing this recombinant host cell under conditions such that saidpolypeptide is expressed and recovering said polypeptide. Also preferredis this method of making an isolated polypeptide, wherein saidrecombinant host cell is a eukaryotic cell and said polypeptide is aprotein comprising an amino acid sequence selected from the groupconsisting of: an amino acid sequence of SEQ ID NO:2, 24, 54, and/or 56wherein Y is an integer set forth in Table I and said position of the“Total AA of ORF” of SEQ ID NO:2, 24, 54, and/or 56 is defined in TableI; and an amino acid sequence of a protein encoded by a cDNA cloneidentified by a cDNA Clone Identifier in Table I and contained in thedeposit with the ATCC Deposit Number shown for said cDNA clone in TableI. The isolated polypeptide produced by this method is also preferred.

Also preferred is a method of treatment of an individual in need of anincreased level of a protein activity, which method comprisesadministering to such an individual a pharmaceutical compositioncomprising an amount of an isolated polypeptide, polynucleotide, orantibody of the claimed invention effective to increase the level ofsaid protein activity in said individual.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

REFERENCES

-   Altschul S F; Gish W; Miller W; Myers E W; Lipman D J Basic local    alignment search tool. J. Mol. Biol. 215:403-10 (1990).-   Bartlett et al. Molecular Recognition in Chemical and Biological    Problems Special Publication, Royal Chem. Soc. 78:182-196 (1989).-   Bohm H-J, LUDI: rule-based automatic design of new substituents for    enzyme inhibitor leads. J. Comp. Aid. Molec. Design 6:61-78 (1992)-   Cardozo T; Totrov M; Abagyan R Homology modeling by the ICM method.    Proteins 23:403-14 (1995).-   Goodford, P. J. A computational procedure for determining    energetically favorable binding sites on biologically important    macromolecules. J. Med. Chem. 28:849-857 (1985)-   Goodsell, D. S. and Olsen, A. J. Automated docking of substrates to    proteins by simulated annealing. Proteins 8:195-202 (1990)-   Greer J Comparative modeling of homologous proteins. Methods Enzymol    202:239-52 (1991).-   Hendlich M; Lackner P; Weitckus S; Floeckner H; Froschauer R;    Gottsbacher K; Casari G; Sippl M J Identification of native protein    folds amongst a large number of incorrect models. The calculation of    low energy conformations from potentials of mean force. J. Mol.    Biol. 216:167-80 (1990).-   Hosfield, C., Elce, J. S., Davies, P., Jia, Z., Crystal structure of    calpain reveals the structural basis for Ca²⁺-dependent protease    activity and a novel mode of enzyme activation., EMBO J., 18,    6880-6889 (1999).-   Kuntz I D, Blaney J M, Oatley S J, Langridge R, Ferrin T E. A    geometric approach to macromolecule-ligand interactions. J. Mol.    Biol. 161:269-288 (1982)-   Lesk, A. M., Boswell, D. R., Homology Modeling: Inferences from    Tables of Aligned Sequences. Curr. Op. Struc. Biol. 2:242-247 (1992)-   Martin, Y. C. 3D database searching in drug design. J. Med. Chem.    35:2145-2154 (1992)-   Pearson W R Rapid and sensitive sequence comparison with FASTP and    FASTA. Methods Enzymol 183:63-98 (1990).-   Sali A; Potterton L; Yuan F; van Vlijmen H; Karplus M Evaluation of    comparative protein modeling by MODELLER. PROTEINS 23:318-26 (1995).-   Strobl, S., Femandez-Catalan, C., Braun, M., Huber, R, Masumoto, H.,    Nakagawa., K, Irie, A., Sorimachi, S., Bourenkow, G., Bartunik, H.,    Suzuki, K, Bode, W., The crystal structure of calcium-free human    mcalpain suggests an electrostatic switch mechanism for activation    by calcium. Proc. Natl. Acad. Sci., 2000, 97(2), 588-592.

EXAMPLES Description of the Preferred Embodiments Example 1Bioinformatics Analysis

To search for novel protease inhibitors, a Hidden-Markov Model (HMM) ofcysteine proteases (obtained from the Pfam database in Sanger center)was used to search against the human genomic sequence database using acomputer program called GENEWISEDB. Genomic sequences that were found tohave a GENEWISEDB matching score of more than 15 were selected forfurther analysis. The genomic sequence contained in BAC (bacteriaartificial chromosome) AC015980 (Genbank Accession No. AC015980) wasfound to contain a putative exon sequence. The portion of the sequencefrom AC015980 that matched was extracted and back-searched against theGenbank non-redundant protein database using the BLASTX program (SEQ IDNO:26). The most similar protein sequence, the human CAN5 protein (SEQID NO:4), was used as a template to predict more exons from AC015980using the GENEWISEDB program (see FIG. 7). The final predicted exonswere assembled and a full length clone of the gene was obtained usingthe predicted exon sequences. The protein sequence was found to havesignificant sequence homology with a family of known proteases. Based onthe sequence, structure and known calpain signature sequences, the novelgene was determined to represent a novel human calpain protease and haveprovisionally named the gene CAN-12 (calcium activated neutral protease12) and the protease itself as calpain 12.

Example 2 Method for Constructing a Size Fractionated Brain and TestiscDNA Library

Brain and testis poly A+ RNA was purchased from Clontech and convertedinto double stranded cDNA using the SuperScript™ Plasmid System for cDNASynthesis and Plasmid Cloning (Life Technologies) except that noradioisotope was incorporated in either of the cDNA synthesis steps andthat the cDNA was fractionated by HPLC. This was accomplished on aTransGenomics HPLC system equipped with a size exclusion column(TosoHass) with dimensions of 7.8 mm×30 cm and a particle size of 10 um.Tris buffered saline was used as the mobile phase and the column was runat a flow rate of 0.5 mL/min.

The resulting chromatograms were analyzed to determine which fractionsshould be pooled to obtain the largest cDNA's; generally fractions thateluted in the range of 12 to 15 minutes were pooled. The cDNA wasprecipitated prior to ligation into the Sal I/Not I sites in the pSportvector supplied with the kit. Using a combination of PCR with primers tothe ends of the vector and Sal I/Not I restriction enzyme digestion ofmini-prep DNA, it was determined that the average insert size of thelibrary was greater the 3.5 Kb. The overall complexity of the librarywas greater that 10⁷ independent clones. The library was amplified insemi-solid agar for 2 days at 30° C. An aliquot (200 microliters) of theamplified library was inoculated into a 200 ml culture forsingle-stranded DNA isolation by super-infection with a f1 helper phage.After overnight growth, the released phage particles with precipitatedwith PEG and the DNA isolated with proteinase K, SDS and phenolextractions. The single stranded circular DNA was concentrated byethanol precipitation and used for the cDNA capture experiments.

Example 3 Method of Cloning the Novel Human CAN-12 Calpain

Using the predicted exon genomic sequence from bac AC015980 (FIG. 7; SEQID NO:13, 14, 15, 16, 17, and 26) an antisense 80 bp oligonucleotidewith biotin on the 5′ end was designed with the following sequence; (SEQID NO:18) 5′bAGGGAGCCACTGCCGATGGAGCTCAGGGTGGCCGGGAAGCTGGTGTCTTCAAAGAGGCAGCCATTCCTCAGGCACTC-3′

One microliter (one hundred and fifty nanograms) of the biotinylatedoligonucleotide was added to six microliters (six micrograms) of amixture of single-stranded covalently closed circular liver, brain,testis, and spleen cDNA libraries and seven microliters of 100%formamide in a 0.5 ml PCR tube. The library was a mixture of the brainand testis cDNA library referenced in Example 2, in addition to,commercially available liver and spleen cDNA libraries from LifeTechnologies (Rockville, Md.). The mixture was heated in a thermalcycler to 95° C. for 2 mins. Fourteen microliters of 2× hybridizationbuffer (50% formamide, 1.5 M NaCl, 0.04 M NaPO₄, pH 7.2, 5 mM EDTA, 0.2%SDS) was added to the heated probe/cDNA library mixture and incubated at42° C. for 26 hours. Hybrids between the biotinylated oligonucleotideand the circular cDNA were isolated by diluting the hybridizationmixture to 220 microliters in a solution containing 1 M NaCl, 10 mMTris-HCl pH 7.5, 1 mM EDTA, pH 8.0 and adding 125 microliters ofstreptavidin magnetic beads. This solution was incubated at 42° C. for60 mins, mixing every 5 mins to resuspend the beads. The beads wereseparated from the solution with a magnet and the beads washed threetimes in 200 microliters of 0.1×SSPE, 0.1% SDS at 45° C.

The single stranded cDNAs were released from the biotinlyatedoligonucleotide/streptavidin magnetic bead complex by adding 50microliters of 0.1 N NaOH and incubating at room temperature for 10mins. Six microliters of 3 M Sodium Acetate was added along with 15micrograms of glycogen and the solution ethanol precipitated with 120microliters of 100% ethanol. The DNA was resuspended in 12 microlitersof TE (10 mM Tris-HCl, pH 8.0), 1 mM EDTA, pH 8.0). The single strandedcDNA was converted into double strands in a thermal cycler by mixing 5microliters of the captured DNA with 1.5 microliters 10 micromolarstandard SP6 primer (homologous to a sequence on the cDNA cloningvector) and 1.5 microliters of 10×PCR buffer. The mixture was heated to95° C. for 20 seconds then ramped down to 59° C. At this time 15microliters of a repair mix, that was preheated to 70° C. (Repair mixcontains 4 microliters of 5 mM dNTPs (1.25 mM each), 1.5 microliters of10×PCR buffer, 9.25 microliters of water, and 0.25 microliters of Taqpolymerase). The solution was ramped back to 73° C. and incubated for 23mins. The repaired DNA was ethanol precipitate and resuspended in 10microliters of TE. Two microliters were electroporated in E. coli DH12Scells and resulting colonies were screen by PCR, using a primer pairdesigned from the genomic exonic sequence to identify the proper cDNAs.

Oligonucleotides used to identity the cDNA by PCR.

-   AC015980-L1 GACTTTGAGGCCCTGCTG (SEQ ID NO: 19)-   AC015980-R1 ACAGGAACCCAGTTCCCATA (SEQ ID NO:20)

A single cDNA clone was positive by PCR. The insert was sized and theclone was sequenced. The sequence of the cDNA clone revealed anunspliced intron, in addition to a three base-pair deletion (SEQ ID NO:1). Further attempts to obtain more clones with this method wereunsuccessful, so an RT-PCR cloning approach was applied.

The nucleotide sequence and the encoded polypeptide for CAN-12containing the unspliced intron and three base-pair deletion is shown inFIGS. 1A-E (SEQ ID NO: 1).

Example 4 Method of Cloning the Novel Human CAN-12 Calpain via RT-PCR

The cDNA was amplified from testis stranded cDNA and spinal cord firststrand cDNA using the following RT-PCR primer pair in a standard PCRreaction (25 ng of DNA template were added to the reaction mixture alongwith each oligonucleotide primer at a final concentration of 0.2 μMeach. The total volume of the reactions was 50 μL). RT-PCRCACCTGCCATGTCTCTGTG SEQ ID NO:21 Sense PRIMER RT-PCRGATTATAACAAGGTGGTGTTGAAGA (SEQ ID NO:22) Antisense PRIMER

The thermal cycling conditions for the PCR were as follows: 96° C.  2minutes Then 45 cycles of: 94° C. 30 seconds 55° C. 30 seconds 72° C.  3minutes Then one cycle of: 72° C. 10 minutes  4° C. hold

The PCR was then subjected to electrophoresis on a 1% agarose gel. Bandson the gel were visualized of the predicted size. The bands were excisedfrom the gel with a razor blade.

The PCR products were then extracted from the agarose gel slice usingthe Qiagen QIAquick™ Gel Extraction kit. Briefly, 3 volumes of buffer QGare added to the gel slice. The mixture is incubated at 50° C. until theagarose is melted. Then one volume of isopropanol is added. The sampleis applied to a QIAquick spin column and centrifuged for 1 minute athigh speed. The DNA binds to the column. The column is washed byapplying 750 μL of buffer PE to and centrifuging for 1 minute. Thecolumn is then dried by spinning for an additional minute at high speed.The DNA is eluted from the column by applying 30 μL of elution buffer(buffer EB), letting the column stand for 1 minute, then centrifugingthe column at high speed for 1 minute. The eluate is collected in amicrocentrifuge tube.

Next, a ‘TA’ cloning procedure was used to insert the amplified fragmentinto a plasmid vector. In order to use the ‘TA’ cloning strategy, thePCR amplicon must have a 3+ ‘A’ overhang which is generated by Taqpolymerase. Since a high fidelity, proofreading enzyme was used for thePCR amplification, the proofreading properties of the enzyme mix causethe ‘A’ overhang to be removed. Therefore, before the ‘TA’ cloning couldbe done, ‘A’ overhangs had to be added to the PCR product. To do this,the PCR product was incubated for 15 minutes at 72° C. in a mixturecontaining 5 units of Taq polymerase, 1×PCR buffer and 0.2 mM dATP (allfrom Roche). The Taq polymerase is from Thermus aquaticus BM,recombinant E. coli. The 10× PCR buffer contains 100 mM Tris-HCl, 15 mMMgCl₂, 500 mM KCl, pH 8.3.

The PCR products with added 3′ ‘A’ overhangs was then immediately usedfor ‘TA’ cloning. To do this, the TOPO TA Cloning® Kit for Sequencingfrom Invitrogen was used.

The following reaction mixture was set up:

-   -   −4 μL PCR product    -   −1 μL Salt Solution    -   −1 μL pCR®4-TOPO® vector        This was incubated at room temperature for 5 minutes.

Then 2 μl of this reaction were added to a vial of TOP10 One Shot®chemically competent E. Coli. This was incubated on ice for 5 minutes.The cells were then heat shocked at 42° C. for 30 seconds. Cells weretransferred to ice for another 5 minutes. 250 μl of room temperatureS.O.C. medium was added to the cells. The cells were then incubated at37° C. for 1 hour with shaking for aeration. 50 μL of cells were spreadon selective plates containing 50 82 g/μL carbenicillin and incubated at37° C. overnight. A more detailed protocol for this kit from Invitrogenis available from their website (http://www.invitrogen.com).

The next step was to screen colonies that grew on the selective platesfor positive clones. This was done by growing 7 colonies from the testisPCR and 7 from the spinal cord PCR overnight in 4 mL of LB brothcontaining 50 μg/μL carbenicillin. The plasmid DNA was then isolatedfrom the bacteria using the Qiagen QIAquick Spin Miniprep Kit. Protocolsfor this are available from the Qiagen company web site(http:H/www.qiagen.com).

Once the plasmid DNA was purified, a restriction digest analysis wasperformed to determine if the clones were correct. A restriction enzymedigestion was performed with NotI and SpeI restriction endonucleases,using the suggested buffer and 5 μl of the purified plasmid, 5 units ofenzyme in a total volume of 20 μl. The mixture was incubated at 37° C.for 2 hours. The digest was visualized by electrophoretic separation ona 1% agarose gel stained with ethidium bromide. From this analysis itwas apparent the plasmids contained one size insert corresponding to thepredicted insert size of the transcript. Two clones from each PCRreaction were sequenced using Applied Biosystems BigDye™ dideoxyterminator cycle sequencing on an Applied Biosystems 3700 capillaryarray DNA sequencer.

The above method resulted in several positive clones. After sequencing,it was determined that all of them had an unspliced intron thatintroduced a stop codon, with the exception of clone 1 e (CAN-12v1;FIGS. 8A-C; SEQ ID NO:53). Clone 1 e was correct, except for it wasmissing 3 amino acids in the middle of the protein (See FIGS. 10A-B). Itis thought that these three amino acids may represent an exon of theCAN-12v1 polynucleotide.

Although the other clones contained the unspliced intron, one of theclones, clone 1 b, had the missing 3 amino acids. Thus, clone 1 e andclone 1 b were recombinately combined together effectively cutting outthe region of clone 1 e that was missing the terminal three amino acids,and replacing it with the same region from clone 1 b which did have thethree amino acids. The resulting recombinant clone is named clone 1 e 1b (CAN-12v2), and is provided in FIGS. 9A-C (SEQ ID NO:55).

The method of recombinately combining clone 1 e and clone 1 b areprovided below. The part of clone 1 e that had the missing exon wasexcised by restriction digestion with unique cutting enzymes BamHI andAval. The corresponding fragment with the small exon (three amino acids)from clone 1 b was excised with the same enzymes and inserted into clone1 e. This yielded the complete correct coding sequence that waspredicted. The clone was called 1 e 1 b (CAN-12v2; FIGS. 9A-C; SEQ IDNO:55). CAN-12v2 is believed to represent the true physioligical form ofCAN-12.

Example 5 Expression Profiling of the Novel Human CAN-12 Calpain

The same PCR primer pair that was used to identify the novel CAN-12 cDNAclones via RT-PCR (SEQ ID NO: 21 and 22) was used to measure the steadystate levels of mRNA by quantitative PCR. Briefly, first strand cDNA wasmade from commercially available mRNA. The relative amount of cDNA usedin each assay was determined by performing a parallel experiment using aprimer pair for a gene expressed in equal amounts in all tissues,cyclophilin. The cyclophilin primer pair detected small variations inthe amount of cDNA in each sample and these data were used fornormalization of the data obtained with the primer pair for the novelCAN-12. The PCR data was converted into a relative assessment of thedifference in transcript abundance amongst the tissues tested and thedata is presented in FIG. 4. Transcripts corresponding to CAN-12 wereexpressed highly in the spinal cord; significantly in lymph node,thymus, and to a lesser extent, in spleen.

Example 6 Method of Assessing the Expression Profile of the Novel CAN-12Polypeptides of the Present Invention using Expanded mRNA Tissue andCell Sources

Total RNA from tissues was isolated using the TriZol protocol(Invitrogen) and quantified by determining its absorbance at 260 nM. Anassessment of the 18s and 28s ribosomal RNA bands was made by denaturinggel electrophoresis to determine RNA integrity.

The specific sequence to be measured was aligned with related genesfound in GenBank to identity regions of significant sequence divergenceto maximize primer and probe specificity. Gene-specific primers andprobes were designed using the ABI primer express software to amplifysmall amplicons (150 base pairs or less) to maximize the likelihood thatthe primers function at 100% efficiency. All primer/probe sequences weresearched against Public Genbank databases to ensure target specificity.Primers and probes were obtained from ABI.

For CAN-12, the primer probe sequences were as follows Forward Primer5′-TCGTGCCCTGCATATTGGA-3′ (SEQ ID NO:143) Reverse Primer5′-AAAAGATGTGCTTCCTGGAGAAGA-3′ (SEQ ID NO:144) TaqMan Probe5′-CCCACCAGAAGTCAGAGTTCGTCCTCAG-3′ (SEQ ID NO:145)DNA Contamination

To access the level of contaminating genomic DNA in the RNA, the RNA wasdivided into 2 aliquots and one half was treated with Rnase-free Dnase(Invitrogen). Samples from both the Dnase-treated and non-treated werethen subjected to reverse transcription reactions with (RT+) and without(RT−) the presence of reverse transcriptase. TaqMan assays were carriedout with gene-specific primers (see above) and the contribution ofgenomic DNA to the signal detected was evaluated by comparing thethreshold cycles obtained with the RT+/RT− non-Dnase treated RNA to thaton the RT+/RT− Dnase treated RNA. The amount of signal contributed bygenomic DNA in the Dnased RT− RNA must be less that 10% of that obtainedwith Dnased RT+ RNA. If not the RNA was not used in actual experiments.

Reverse Transcription reaction and Sequence Detection

100 ng of Dnase-treated total RNA was annealed to 2.5 μM of therespective gene-specific reverse primer in the presence of 5.5 mMMagnesium Chloride by heating the sample to 72° C. for 2 min and thencooling to 55° C. for 30 min. 1.25 U/μl of MuLv reverse transcriptaseand 500 μM of each DNTP was added to the reaction and the tube wasincubated at 37° C. for 30 min. The sample was then heated to 90° C. for5 min to denature enzyme.

Quantitative sequence detection was carried out on an ABI PRISM 7700 byadding to the reverse transcribed reaction 2.5 μM forward and reverseprimers, 500 μM of each dNTP, buffer and 5U AmpliTaq Gold™. The PCRreaction was then held at 94° C. for 12 min, followed by 40 cycles of94° C. for 15 sec and 60° C. for 30 sec.

Data Handling

The threshold cycle (Ct) of the lowest expressing tissue (the highest Ctvalue) was used as the baseline of expression and all other tissues wereexpressed as the relative abundance to that tissue by calculating thedifference in Ct value between the baseline and the other tissues andusing it as the exponent in 2^((ΔCt))

The expanded expression profile of the CAN-12 polypeptide, is providedin FIG. 12 and described elsewhere herein.

Example 7 Method of Measuring the Protease Activity of CAN-12Polypeptides

Protease activity of the CAN-12 polypeptide are measured by followingthe inhibition of proteolytic activity in cells, tissues, and/or in invitro assays. Cysteine proteases of the calpain family (of which thepresent invention is a member) catalyze the hydrolysis of peptide,amide, ester, thiol ester and thiono ester bonds. Any assay thatmeasures cleavage of these bonds can be used to quantitate enzymaticactivity. In vitro assays for measuring protease activity usingsynthetic peptide fluorescent, spectrophotometric either through the useof single substrates (see below for examples), and fluorescenceresonance transfer assays are well described in the art, as singlesubstrates or as part of substrate libraries (Backes et al., 2000;Knight, C. G. Fluorimetric Assays of Proteolytic Enzymes. Meth. Enzymol.248: 18-34 (1995)). In addition proteolytic activity is measured byfollowing production of peptide products. Such approaches are well knownto those familiar with the art (reviewed in McGeehan, G. M., Bickett, D.M., Wiseman, J. S., Green, M., Berman, Meth. Enzymol. 248: 35-46 (1995))

A complete set of protocols that have been used to evaluate calpainactivity and are provided in Calpain Methods and Protocols John Elce ed.In Meth. Mol. Biol. Volume 144, 2000 (Humana Press, Totowa, N.J.).

Inhibitor Identification

Early work on calpain inhibitors produced nonselective enzymeinhibitors. Peptidyl aldehydes such as leupeptin and antipain inhibitcalpain but also other proteases including serine proteases.Irreversible inhibitors such as the E64 family have also been studied,and peptidyl halomethanes and diazomethanes have long been used asprotease inhibitors (Hayes et al., Drug News Perspect 11:215-222, 1998).Given the multiple therapeutic indications for the inhibition of calpainit appears that the achievement of selective modulators includingspecific inhibitors of this enzyme is an important goal.

The CAN-12 may be incubated with potential inhibitors (preferably smallmolecule inhibitors or antibodies provided elsewhere herein) fordifferent times and with varying concentrations. Residual proteaseactivity could then be measured according to any appropriate means knownin the art. Enzyme activity in the presence of control may be expressedas fraction of control and curve fit to pre-incubation time and serpinconcentration to determine inhibitory parameters including concentrationthat half maximally inhibits the enzyme activity.

Non-limiting examples of in vitro protease assays are well described inthe art. Non-limiting examples of a spectrophotometric protease assaysare the thrombin and tryptase assays measuring time-dependent opticaldensity change followed at 405 nm using a kinetic microplate reader(Molecular Devices UVmax)(Balasubramanian, et al., Active site-directedsynthetic thrombin inhibitors: synthesis, in vitro and in vivo activityprofile of BMY 44621 and analogs an examination of the role of the aminogroup in the D-Phe-Pro-Arg-H series. J. Med. Chem. 36:300-303 (1993);and Combrink et al., Novel 1,2-Benzisothiazol-3-one-1,1-dioxideInhibitors of Human Mast Cell Tryptase. J. Med. Chem. 41:4854-4860(1998)).

An example of a fluorescence assay which m-ay be used for the presentinvention is the Factor VIIa assay. Briefly, the Factor VIIa assay ismeasured in the presence of human recombinant tissue factor (INNOVINfrom Dade Behring Cat.# B4212-100). Human Factor VIIa may be obtainedfrom Enzyme Research Labs (Cat.# HFVIIA 1640). Enzymatic activity couldbe measured in a buffer containing 150 mM NaCl, 5 mM CaCl₂, 1 mM CHAPSand 1 mg/ml PEG 6000 (pH 7.4) with 1 nM FVIIa and 100 μMD-Ile-Pro-Arg-AFC (Enzyme Systems Products, Km>200 ) 0.66% DMSO. Theassay (302 μl total volume) may be incubated at room temperature for 2hr prior to reading fluorometric signal (Ex 405/Em 535) using aMolecular Devices or Victor 2 (Wallac) fluorescent plate reader.

In addition to the methods described above, protease activity (andtherefore serpin activity) can be measured using fluorescent resonanceenergy transfer (FRET with Quencher -P_(n)-P₃-P₂-P₁--P₁′-P₂′-Fluorophore), fluorescent peptide bound to beads (Fluorophore-P_(n)-P₃- P₂-P₁- -P₁′-P₂′-Bead), dye-protein substrates andserpin-protease gel shifts. All of which are well known to those skilledin the art (see a non-limiting review in Knight, C. G. FluorimetricAssays of Proteolytic Enzymes. Meth. Enzymol. 248: 18-34 (1995)).

Additional assay methods are known in the art and are encompassed by thepresent invention. See, for example, Backes B J, Harris J L, Leonetti F,Craik C S, Ellman J A. Synthesis of positional-scanning libraries offluorogenic peptide substrates to define the extended substratespecificity of plasmin and thrombin. Nat Biotechnol. 18:187-93 (2000);Balasubramanian, et al., Active site-directed synthetic thrombininhibitors: synthesis, in vitro and in vivo activity profile of BMY44621 and analogs an examination of the role of the amino group in theD-Phe-Pro-Arg-H series. J. Med. Chem. 36:300-303 (1993); and Combrink etal., Novel 1,2-Benzisothiazol-3-one-1,1-dioxide Inhibitors of Human MastCell Tryptase. J. Med. Chem. 41:4854-4860 (1998) and those methodsdescribed in: Calpain Methods and Protocols (ed J. S. Elce) Meth. Mol.Biol. 144, 2000 and Calpain: Pharmacology and Toxicology of acalcium-dependent protease (K. Wang & P.-W. Yuen editors) Taylor &Francis Philadelphia, Pa., 1999; which are hereby incorporated herein byreference in their entirety.

Example 8 Determination of the Preferred Substrate Sequence of theCAN-12 Protease

The preferred substrate sequence specificity of the CAN-12metalliprotease of the present invention may be determined using usingtwo redundant peptide libraries and Edman peptide sequencing (1-2) asshown below.

The first peptide library is random, can vary in length and incorporatesa modification at the N-terminus to block Edman sequencing. In theexample provided, biotin is used as the blocking group. Proteolyticcleavage of the library is allowed to proceed long enough to turn overapproximately 5-10% of the library. Edman sequencing of the peptidemixture provides the preferred substrate residues for the P′ sites onthe protease. The second peptide library has fixed P′ residues torestrict the proteolytic cleavage site and an affinity tag for removingthe C-terminal product of the proteolysis, leaving the N-terminalpeptide product pool behind for Edman sequencing to determine the aminoacid residues preferred in the P1, P2, P3 etc. sites of the protease.

Reagents.

The endoproteases Factor Xa (New England BioLabs, Inc., Beverly, Mass.)and human kidney Renin (Calbiochem, San Diego, Calif.) were purchasedfor validation experiments. A hexapeptide library containing 4.7×10⁷peptide species was synthesized by the Molecular Redesign group(Natarajan & Riexinger) at Bristol-Myers Squibb Company (Princeton,N.J.). The library contained equivalent representation of 19 amino acidresidues at each of the six degenerate positions and incorporated anN-terminal biotin group and a C-terminal amide. Cysteine residues wereexcluded from the peptide pool and Methionine residues were replacedwith Norleucine.

Endoprotease Cleavage of the Peptide Library.

The following method may be used to determine the preferred substratesequence downstream of the cleavage site. A 1.88 mM peptide librarysolution is prepared in phosphate buffer (10 mM Sodium Phosphate (pH7.6), 0.1 M NaCl, and 10% DMSO) and is incubated with 2-30 μgendoprotease at 37° C. Using a fluorescamine assay to estimate theextent of peptide cleavage, the reaction is stopped at 5-10% completionwith incubation at 100° C. for 2.0 minutes. Peptide pools are subjectedto Edman sequencing. The data obtained is normalized and corrected fordifferences in efficiency of cleavage and recovery in the sequencer.

Fluorescamine Assay to Monitor Peptide Cleavage.

Primary amines generated during peptide cleavage is measured by reactionwith fluorescamine (Aldrich, St. Louis, Mo.), as described in reference3. The relative fluorescence is determined by measuring signals at□^(ex)=355 nm and □^(em)=460 nm on a PerkinElmer Wallac 1420Spectrofluorometer. Reactions are sampled at multiple time points andassayed in triplicate. The amount of cleavage product formed isdetermined using the relative fluorescence produced by varyingconcentrations of a peptide standard of known concentration.

References

-   (1) “Substrate Specificity of Cathepsins D and E Determined by    N-Terminal and C-Terminal Sequencing of Peptide Pools” D. Arnold et    al. (1997) Eur. J. Biochem. 249, 171.-   (2) “Determination of Protease Cleavage Site Motifs Using    Mixture-Based Oriented Peptide Libraries” B. E. Turk et al. (2001)    Nature Biotech. 19, 661.-   (3) “Fluorescamine: a Reagent for Assay of Amino Acids, Peptides,    Proteins, and Primary Amines in the Picomole Range” S.    Udenfirend, S. Stein, P. Bohlen, W. Dairman, W. Leimgruber, and M.    Weigele (1972) Science 178, 87.

Example 9 Chromosomal Mapping of Calpain 12 and Linkage toNeurodegenerative Disorders

The calpain 12 polynucleotides of the present invention were used todetermine the chromosomal localization of the calpain 12 gene. Thecomparison of the chromosomal location of the calpain 12 gene with thelocation of chromosomal regions which have been shown to be associatedwith specific diseases or conditions, e.g. by linkage analysis, can beindicative of diseases in which calpain 12 may play a role.

A chromosomal localization of the calpain 12 gene was performed by usingthe nucleic acid sequence (SEQ ID NO:1) of the invention in a databasesearch of the recently completed draft of human genome sequence. Usingthe Basic Local Alignment Search Tool 2 (BLAST2), the first 200 bp ofcalpain 12 cDNA sequence showed a perfect alignment of nucleotides 62 to200 with the minus strand of Homo sapiens chromosome 2 clone RP11-541A15nucleotide 177952 to 177814, and a perfect alignment of nucleotide 1 to62 of calpain 12 cDNA (SEQ ID NO:1) with the nucleotide 94118 to 94179of the same clone, suggesting the likelihood of an intron interveningthis 200 cDNA fragment. To confirm the map of calpain 12 gene, anotherBLAST2 search was done with a 300 pb fragment of calpain 12 cDNAsequence containing nucleotides 4251 up to poly A signal sequence atposition 4550 (SEQ ID NO:1). An alignment with 97% identities (292/300)of this calpain 3′ sequence was found with nucleotide 125724 to 126023of the same Homo sapiens chromosome 2 clone RP11-541A15. In order to geta refined map of this clone containing the locus of calpain 12, a mapsearch of NCBI genome database was performed. RP11-541A15 (Acc#AC015980.2) was found within a BAC contig adjacent to clone 852C13(AL133246.2) mapping in 2p21-p22.

A whole-genome linkage scan in multiple sclerosis families (Ebers et al.A full genome search in multiple sclerosis. Nature Genet. 13: 472-476,1996.) identified 5 susceptibility loci on chromosomes 2, 3, 5, 11, andX. In particular, an association was identified with marker D2S119 onchromosome 2 and MS. We further delineated the localization of thismarker, D2S119, on 2p16-p21 based on a radiation hybrid linkage mapretrieved from an online query at NCBI web site:http:/www.ncbi.nlm.nih.gov/genome/sts/. Since the map of calpain 12 andthe susceptibility marker D2S119 overlaps, it is reasonable to postulatethat calpain 12 may contribute to MS. Furthermore, the transcriptionprofile of calpain 12 indicated a prominent expression in spinal cord,and implication of calpains in MS has been suggested (Shields D C et al.A putative mechanism of demyelination in multiple sclerosis by aproteolytic enzyme, calpain. Proc Natl Acad Sci USA. 96:11486-91.1999).

Example 10 Method of Screening for Compounds that Interact with theCAN-12 Polypeptide

The following assays are designed to identify compounds that bind to theCAN-12 polypeptide, bind to other cellular proteins that interact withthe CAN-12 polypeptide, and to compounds that interfere with theinteraction of the CAN-12 polypeptide with other cellular proteins.

Such compounds can include, but are not limited to, other cellularproteins. Specifically, such compounds can include, but are not limitedto, peptides, such as, for example, soluble peptides, including, but notlimited to Ig-tailed fusion peptides, comprising extracellular portionsof CAN-12 polypeptide transmembrane receptors, and members of randompeptide libraries (see, e.g., Lam, K. S. et al., 1991, Nature 354:82-84;Houghton, R. et al., 1991, Nature 354:84-86), made of D- and/orL-configuration amino acids, phosphopeptides (including, but not limitedto, members of random or partially degenerate phosphopeptide libraries;see, e.g., Songyang, Z., et al., 1993, Cell 72:767-778), antibodies(including, but not limited to, polyclonal, monoclonal, humanized,anti-idiotypic, chimeric or single chain antibodies, and FAb,F(ab′).sub.2 and FAb. expression libary fragments, and epitope-bindingfragments thereof), and small organic or inorganic molecules.

Compounds identified via assays such as those described herein can beuseful, for example, in elaborating the biological function of theCAN-12 polypeptide, and for ameliorating symptoms of tumor progression,for example. In instances, for example, whereby a tumor progressionstate or disorder results from a lower overall level of CAN-12expression, CAN-12 polypeptide, and/or CAN-12 polypeptide activity in acell involved in the tumor progression state or disorder, compounds thatinteract with the CAN-12 polypeptide can include ones which accentuateor amplify the activity of the bound CAN-12 polypeptide. Such compoundswould bring about an effective increase in the level of CAN-12polypeptide activity, thus ameliorating symptoms of the tumorprogression disorder or state. In instances whereby mutations within theCAN-12 polypeptide cause aberrant CAN-12 polypeptides to be made whichhave a deleterious effect that leads to tumor progression, compoundsthat bind CAN-12 polypeptide can be identified that inhibit the activityof the bound CAN-12 polypeptide. Assays for testing the effectiveness ofsuch compounds are known in the art and discussed, elsewhere herein.

Example 11 Method of Screening, In Vitro, Compounds that Bind to theCAN-12 Polypeptide

In vitro systems can be designed to identify compounds capable ofbinding the CAN-12 polypeptide of the invention. Compounds identifiedcan be useful, for example, in modulating the activity of wild typeand/or mutant CAN-12 polypeptide, preferably mutant CAN-12 polypeptide,can be useful in elaborating the biological function of the CAN-12polypeptide, can be utilized in screens for identifying compounds thatdisrupt normal CAN-12 polypeptide interactions, or can in themselvesdisrupt such interactions.

The principle of the assays used to identify compounds that bind to theCAN-12 polypeptide involves preparing a reaction mixture of the CAN-12polypeptide and the test compound under conditions and for a timesufficient to allow the two components to interact and bind, thusforming a complex which can be removed and/or detected in the reactionmixture. These assays can be conducted in a variety of ways. Forexample, one method to conduct such an assay would involve anchoringCAN-12 polypeptide or the test substance onto a solid phase anddetecting CAN-12 polypeptide /test compound complexes anchored on thesolid phase at the end of the reaction. In one embodiment of such amethod, the CAN-12 polypeptide can be anchored onto a solid surface, andthe test compound, which is not anchored, can be labeled, eitherdirectly or indirectly.

In practice, microtitre plates can conveniently be utilized as the solidphase. The anchored component can be immobilized by non-covalent orcovalent attachments. Non-covalent attachment can be accomplished bysimply coating the solid surface with a solution of the protein anddrying. Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific for the protein to be immobilized can be used toanchor the protein to the solid surface. The surfaces can be prepared inadvance and stored.

In order to conduct the assay, the nonimmobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslyimmobilized component is pre-labeled, the detection of label immobilizedon the surface indicates that complexes were formed. Where thepreviously nonimmobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the immobilized component (theantibody, in turn, can be directly labeled or indirectly labeled with alabeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for CAN-12polypeptide or the test compound to anchor any complexes formed insolution, and a labeled antibody specific for the other component of thepossible complex to detect anchored complexes.

Example 12 Method of Identifying Compounds that Interfere with CAN-12Polypeptide/Cellular Product Interaction

The CAN-12 polypeptide of the invention can, in vivo, interact with oneor more cellular or extracellular macromolecules, such as proteins. Suchmacromolecules include, but are not limited to, nucleic acid moleculesand those products identified via methods such as those described,elsewhere herein. For the purposes of this discussion, such cellular andextracellular macromolecules are referred to herein as “bindingpartner(s)”. For the purpose of the present invention, “binding partner”may also encompass small molecule compounds, polysaccarides, lipids, andany other molecule or molecule type referenced herein. Compounds thatdisrupt such interactions can be useful in regulating the activity ofthe CAN-12 polypeptide, especially mutant CAN-12 polypeptide. Suchcompounds can include, but are not limited to molecules such asantibodies, peptides, and the like described in elsewhere herein.

The basic principle of the assay systems used to identify compounds thatinterfere with the interaction between the CAN-12 polypeptide and itscellular or extracellular binding partner or partners involves preparinga reaction mixture containing the CAN-12 polypeptide, and the bindingpartner under conditions and for a time sufficient to allow the twoproducts to interact and bind, thus forming a complex. In order to testa compound for inhibitory activity, the reaction mixture is prepared inthe presence and absence of the test compound. The test compound can beinitially included in the reaction mixture, or can be added at a timesubsequent to the addition of CAN-12 polypeptide and its cellular orextracellular binding partner. Control reaction mixtures are incubatedwithout the test compound or with a placebo. The formation of anycomplexes between the CAN-12 polypeptide and the cellular orextracellular binding partner is then detected. The formation of acomplex in the control reaction, but not in the reaction mixturecontaining the test compound, indicates that the compound interfereswith the interaction of the CAN-12 polypeptide and the interactivebinding partner. Additionally, complex formation within reactionmixtures containing the test compound and normal CAN-12 polypeptide canalso be compared to complex formation within reaction mixturescontaining the test compound-and mutant CAN-12 polypeptide. Thiscomparison can be important in those cases wherein it is desirable toidentify compounds that disrupt interactions of mutant but not normalCAN-12 polypeptide.

The assay for compounds that interfere with the interaction of theCAN-12 polypeptide and binding partners can be conducted in aheterogeneous or homogeneous format. Heterogeneous assays involveanchoring either the CAN-12 polypeptide or the binding partner onto asolid phase and detecting complexes anchored on the solid phase at theend of the reaction. In homogeneous assays, the entire reaction iscarried out in a liquid phase. In either approach, the order of additionof reactants can be varied to obtain different information about thecompounds being tested. For example, test compounds that interfere withthe interaction between the CAN-12 polypeptide and the binding partners,e.g., by competition, can be identified by conducting the reaction inthe presence of the test substance; i.e., by adding the test substanceto the reaction mixture prior to or simultaneously with the CAN-12polypeptide and interactive cellular or extracellular binding partner.Alternatively, test compounds that disrupt preformed complexes, e.g.compounds with higher binding constants that displace one of thecomponents from the complex, can be tested by adding the test compoundto the reaction mixture after complexes have been formed. The variousformats are described briefly below.

In a heterogeneous assay system, either the CAN-12 polypeptide or theinteractive cellular or extracellular binding partner, is anchored ontoa solid surface, while the non-anchored species is labeled, eitherdirectly or indirectly. In practice, microtitre plates are convenientlyutilized. The anchored species can be immobilized by non-covalent orcovalent attachments. Non-covalent attachment can be accomplished simplyby coating the solid surface with a solution of the CAN-12 polypeptideor binding partner and drying. Alternatively, an immobilized antibodyspecific for the species to be anchored can be used to anchor thespecies to the solid surface. The surfaces can be prepared in advanceand stored.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. The detection of complexes anchored on the solid surface can beaccomplished in a number of ways. Where the non-immobilized species ispre-labeled, the detection of label immobilized on the surface indicatesthat complexes were formed. Where the non-immobilized species is notpre-labeled, an indirect label can be used to detect complexes anchoredon the surface; e.g., using a labeled antibody specific for theinitially non-immobilized species (the antibody, in turn, can bedirectly labeled or indirectly labeled with a labeled anti-Ig antibody).Depending upon the order of addition of reaction components, testcompounds which inhibit complex formation or which disrupt preformedcomplexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds which inhibit complex or which disrupt preformed complexes canbe identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. In this approach, a preformed complex of the CAN-12 polypeptideand the interactive cellular or extracellular binding partner product isprepared in which either the CAN-12 polypeptide or their bindingpartners are labeled, but the signal generated by the label is quencheddue to complex formation (see, e.g., U.S. Pat. No. 4,109,496 byRubenstein which utilizes this approach for immunoassays). The additionof a test substance that competes with and displaces one of the speciesfrom the preformed complex will result in the generation of a signalabove background. In this way, test substances which disrupt CAN-12polypeptide -cellular or extracellular binding partner interaction canbe identified.

In a particular embodiment, the CAN-12 polypeptide can be prepared forimmobilization using recombinant DNA techniques known in the art. Forexample, the CAN-12 polypeptide coding region can be fused to aglutathione-S-transferase (GST) gene using a fusion vector such aspGEX-5×-1, in such a manner that its binding activity is maintained inthe resulting fusion product. The interactive cellular or extracellularproduct can be purified and used to raise a monoclonal antibody, usingmethods routinely practiced in the art and described above. Thisantibody can be labeled with the radioactive isotope sup. 125 I, forexample, by methods routinely practiced in the art. In a heterogeneousassay, e.g., the GST-CAN-12 polypeptide fusion product can be anchoredto glutathione-agarose beads. The interactive cellular or extracellularbinding partner product can then be added in the presence or absence ofthe test compound in a manner that allows interaction and binding tooccur. At the end of the reaction period, unbound material can be washedaway, and the labeled monoclonal antibody can be added to the system andallowed to bind to the complexed components. The interaction between theCAN-12 polypeptide and the interactive cellular or extracellular bindingpartner can be detected by measuring the amount of radioactivity thatremains associated with the glutathione-agarose beads. A successfulinhibition of the interaction by the test compound will result in adecrease in measured radioactivity.

Alternatively, the GST-CAN-12 polypeptide fusion product and theinteractive cellular or extracellular binding partner product can bemixed together in liquid in the absence of the solid glutathione-agarosebeads. The test compound can be added either during or after the bindingpartners are allowed to interact. This mixture can then be added to theglutathione-agarose beads and unbound material is washed away. Again theextent of inhibition of the binding partner interaction can be detectedby adding the labeled antibody and measuring the radioactivityassociated with the beads.

In another embodiment of the invention, these same techniques can beemployed using peptide fragments that correspond to the binding domainsof the CAN-12 polypeptide product and the interactive cellular orextracellular binding partner (in case where the binding partner is aproduct), in place of one or both of the full length products.

Any number of methods routinely practiced in the art can be used toidentify and isolate the protein's binding site. These methods include,but are not limited to, mutagenesis of one of the genes encoding one ofthe products and screening for disruption of binding in aco-immunoprecipitation assay. Compensating mutations in the geneencoding the second species in the complex can be selected. Sequenceanalysis of the genes encoding the respective products will reveal themutations that correspond to the region of the product involved ininteractive binding. Alternatively, one product can be anchored to asolid surface using methods described in this Section above, and allowedto interact with and bind to its labeled binding partner, which has beentreated with a proteolytic enzyme, such as trypsin. After washing, ashort, labeled peptide comprising the binding domain can remainassociated with the solid material, which can be isolated and identifiedby amino acid sequencing. Also, once the gene coding for the cellular orextracellular binding partner product is obtained, short gene segmentscan be engineered to express peptide fragments of the product, which canthen be tested for binding activity and purified or synthesized.

Example 13 Isolation of a Specific Clone from the Deposited Sample

The deposited material in the sample assigned the ATCC Deposit Numbercited in Table I for any given cDNA clone also may contain one or moreadditional plasmids, each comprising a cDNA clone different from thatgiven clone. Thus, deposits sharing the same ATCC Deposit Number containat least a plasmid for each cDNA clone identified in Table I. Typically,each ATCC deposit sample cited in Table I comprises a mixture ofapproximately equal amounts (by weight) of about 1-10 plasmid DNAs, eachcontaining a different cDNA clone and/or partial cDNA clone; but such adeposit sample may include plasmids for more or less than 2 cDNA clones.

Two approaches can be used to isolate a particular clone from thedeposited sample of plasmid DNA(s) cited for that clone in Table I.First, a plasmid is directly isolated by screening the clones using apolynucleotide probe corresponding to SEQ ID NO: 1.

Particularly, a specific polynucleotide with 3040 nucleotides issynthesized using an Applied Biosystems DNA synthesizer according to thesequence reported. The oligonucleotide is labeled, for instance, with32P-ATP using T4 polynucleotide kinase and purified according to routinemethods. (E.g., Maniatis et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Press, Cold Spring, N.Y. (1982).) The plasmid mixtureis transformed into a suitable host, as indicated above (such as XL-1Blue (Stratagene)) using techniques known to those of skill in the art,such as those provided by the vector supplier or in related publicationsor patents cited above. The transformants are plated on 1.5% agar plates(containing the appropriate selection agent, e.g., ampicillin) to adensity of about 150 transformants (colonies) per plate. These platesare screened using Nylon membranes according to routine methods forbacterial colony screening (e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Edit., (1989), Cold Spring Harbor LaboratoryPress, pages 1.93 to 1.104), or other techniques known to those of skillin the art.

Alternatively, two primers of 17-20 nucleotides derived from both endsof the SEQ ID NO: 1 (i.e., within the region of SEQ ID NO: 1 bounded bythe 5′ NT and the 3 ′ NT of the clone defined in Table I) aresynthesized and used to amplify the desired cDNA using the depositedcDNA plasmid as a template. The polymerase chain reaction is carried outunder routine conditions, for instance, in 25 ul of reaction mixturewith 0.5 ug of the above cDNA template. A convenient reaction mixture is1.5-5 mM MgCl2, 0.01% (w/v) gelatin, 20 uM each of DATP, dCTP, dGTP,dTTP, 25 μmol of each primer and 0.25 Unit of Taq polymerase. Thirtyfive cycles of PCR (denaturation at 94 degree C. for 1 min; annealing at55 degree C. for 1 min; elongation at 72 degree C. for 1 min) areperformed with a Perkin-Elmer Cetus automated thermal cycler. Theamplified product is analyzed by agarose gel electrophoresis and the DNAband with expected molecular weight is excised and purified. The PCRproduct is verified to be the selected sequence by subcloning andsequencing the DNA product.

The polynucleotide(s) of the present invention, the polynucleotideencoding the polypeptide of the present invention, or the polypeptideencoded by the deposited clone may represent partial, or incompleteversions of the complete coding region (i.e., full-length gene). Severalmethods are known in the art for the identification of the 5′ or 3,non-coding and/or coding portions of a gene which may not be present inthe deposited clone. The methods that follow are exemplary and shouldnot be construed as limiting the scope of the invention. These methodsinclude but are not limited to, filter probing, clone enrichment usingspecific probes, and protocols similar or identical to 5′ and 3, “RACE”protocols that are well known in the art. For instance, a method similarto 5′ RACE is available for generating the missing 5′ end of a desiredfull-length transcript. (Fromont-Racine et al., Nucleic Acids Res.21(7):1683-1684 (1993)).

Briefly, a specific RNA oligonucleotide is ligated to the 5, ends of apopulation of RNA presumably containing full-length gene RNAtranscripts. A primer set containing a primer specific to the ligatedRNA oligonucleotide and a primer specific to a known sequence of thegene of interest is used to PCR amplify the 5′ portion of the desiredfull-length gene. This amplified product may then be sequenced and usedto generate the full-length gene.

This above method starts with total RNA isolated from the desiredsource, although poly-A+ RNA can be used. The RNA preparation can thenbe treated with phosphatase if necessary to eliminate 5′ phosphategroups on degraded or damaged RNA that may interfere with the later RNAligase step. The phosphatase should then be inactivated and the RNAtreated with tobacco acid pyrophosphatase in order to remove the capstructure present at the 5′ ends of messenger RNAs. This reaction leavesa 5′ phosphate group at the 5′ end of the cap cleaved RNA which can thenbe ligated to an RNA oligonucleotide using T4 RNA ligase.

This modified RNA preparation is used as a template for first strandcDNA synthesis using a gene specific oligonucleotide. The first strandsynthesis reaction is used as a template for PCR amplification of thedesired 5′ end using a primer specific to the ligated RNAoligonucleotide and a primer specific to the known sequence of the geneof interest. The resultant product is then sequenced and analyzed toconfirm that the 5′ end sequence belongs to the desired gene. Moreover,it may be advantageous to optimize the RACE protocol to increase theprobability of isolating additional 5′ or 3′ coding or non-codingsequences. Various methods of optimizing a RACE protocol are known inthe art, though a detailed description summarizing these methods can befound in B. C. Schaefer, Anal. Biochem., 227:255-273, (1995).

An alternative method for carrying out 5′ or 3′ RACE for theidentification of coding or non-coding sequences is provided by Frohman,M. A., et al., Proc. Nat'l. Acad. Sci. USA, 85:8998-9002 (1988).Briefly, a cDNA clone missing either the 5′ or 3′ end can bereconstructed to include the absent base pairs extending to thetranslational start or stop codon, respectively. In some cases, cDNAsare missing the start of translation, therefor. The following brieflydescribes a modification of this original 5′ RACE procedure. Poly A+ortotal RNAs reverse transcribed with Superscript II (Gibco/BRL) and anantisense or I complementary primer specific to the cDNA sequence. Theprimer is removed from the reaction with a Microcon Concentrator(Amicon). The first-strand cDNA is then tailed with dATP and terminaldeoxynucleotide transferase (Gibco/BRL). Thus, an anchor sequence isproduced which is needed for PCR amplification. The second strand issynthesized from the da-tail in PCR buffer, Taq DNA polymerase(Perkin-Elmer Cetus), an oligo-dT primer containing three adjacentrestriction sites (XhoIJ Sail and ClaI) at the 5′ end and a primercontaining just these restriction sites. This double-stranded cDNA isPCR amplified for 40 cycles with the same primers as well as a nestedcDNA-specific antisense primer. The PCR products are size-separated onan ethidium bromide-agarose gel and the region of gel containing cDNAproducts the predicted size of missing protein-coding DNA is removed.cDNA is purified from the agarose with the Magic PCR Prep kit (Promega),restriction digested with XhoI or SalI, and ligated to a plasmid such aspBluescript SKII (Stratagene) at XhoI and EcoRV sites. This DNA istransformed into bacteria and the plasmid clones sequenced to identifythe correct protein-coding inserts. Correct 5′ ends are confirmed bycomparing this sequence with the putatively identified homologue andoverlap with the partial cDNA clone. Similar methods known in the artand/or commercial kits are used to amplify and recover 3′ ends.

Several quality-controlled kits are commercially available for purchase.Similar reagents and methods to those above are supplied in kit formfrom Gibco/BRL for both 5′ and 3′ RACE for recovery of full lengthgenes. A second kit is available from Clontech which is a modificationof a related technique, SLIC (single-stranded ligation tosingle-stranded cDNA), developed by Dumas et al., Nucleic Acids Res.,19:5227-32(1991). The major differences in procedure are that the RNA isalkaline hydrolyzed after reverse transcription and RNA ligase is usedto join a restriction site-containing anchor primer to the first-strandcDNA. This obviates the necessity for the dA-tailing reaction whichresults in a polyT stretch that is difficult to sequence past.

An alternative to generating 5′ or 3′ cDNA from RNA is to use cDNAlibrary double-stranded DNA. An asymmetric PCR-amplified antisense cDNAstrand is synthesized with an antisense cDNA-specific primer and aplasmid-anchored primer. These primers are removed and a symmetric PCRreaction is performed with a nested cDNA-specific antisense primer andthe plasmid-anchored primer.

RNA Ligase Protocol for Generating the 5′ or 3′ End Sequences to ObtainFull Length Genes

Once a gene of interest is identified, several methods are available forthe identification of the 5′ or 3′ portions of the gene which may not bepresent in the original cDNA plasmid. These methods include, but are notlimited to, filter probing, clone enrichment using specific probes andprotocols similar and identical to 5′ and 3′ RACE. While the full-lengthgene may be present in the library and can be identified by probing, auseful method for generating the 5′ or 3′ end is to use the existingsequence information from the original cDNA to generate the missinginformation. A method similar to 5′ RACE is available for generating themissing 5′ end of a desired full-length gene. (This method was publishedby Fromont-Racine et al., Nucleic Acids Res., 21(7): 1683-1684 (1993)).Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of apopulation of RNA presumably 30 containing full-length gene RNAtranscript and a primer set containing a primer specific to the ligatedRNA oligonucleotide and a primer specific to a known sequence of thegene of interest, is used to PCR amplify the 5′ portion of the desiredfull length gene which may then be sequenced and used to generate thefull length gene. This method starts with total RNA isolated from thedesired source, poly A RNA may be used but is not a prerequisite forthis procedure. The RNA preparation may then be treated with phosphataseif necessary to eliminate 5′ phosphate groups on degraded or damaged RNAwhich may interfere with the later RNA ligase step. The phosphatase ifused is then inactivated and the RNA is treated with tobacco acidpyrophosphatase in order to remove the cap structure present at the 5′ends of messenger RNAs. This reaction leaves a 5′ phosphate group at the5′ end of the cap cleaved RNA which can then be ligated to an RNAoligonucleotide using T4 RNA ligase. This modified RNA preparation canthen be used as a template for first strand cDNA synthesis using a genespecific oligonucleotide. The first strand synthesis reaction can thenbe used as a template for PCR amplification of the desired 5′ end usinga primer specific to the ligated RNA oligonucleotide and a primerspecific to the known sequence of the apoptosis related of interest. Theresultant product is then sequenced and analyzed to confirm that the 5′end sequence belongs to the relevant apoptosis related.

Example 14 Bacterial Expression of a Polypeptide

A polynucleotide encoding a polypeptide of the present invention isamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ ends of the DNA sequence, as outlined in Example 13, to synthesizeinsertion fragments. The primers used to amplify the cDNA insert shouldpreferably contain restriction sites, such as BamHI and XbaI, at the 5′end of the primers in order to clone the amplified product into theexpression vector. For example, BamHI and XbaI correspond to therestriction enzyme sites on the bacterial expression vector pQE-9.(Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodesantibiotic resistance (Ampr), a bacterial origin of replication (ori),an IPTG-regulatable promoter/operator (P/O), a ribosome binding site(RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.

The pQE-9 vector is digested with BamHI and XbaI and the amplifiedfragment is ligated into the pQE-9 vector maintaining the reading frameinitiated at the bacterial RBS. The ligation mixture is then used totransform the E. coli strain M15/rep4 (Qiagen, Inc.) which containsmultiple copies of the plasmid pREP4, that expresses the lacI repressorand also confers kanamycin resistance (Kanr). Transformants areidentified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis.

Clones containing the desired constructs are grown overnight (O/N) inliquid culture in LB media supplemented with both Amp (100 ug/ml) andKan (25 ug/ml). The O/N culture is used to inoculate a large culture ata ratio of 1:100 to 1:250. The cells are grown to an optical density 600(O.D.600) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalactopyranoside) is then added to a final concentration of 1 mM. IPTG inducesby inactivating the lacI repressor, clearing the P/O leading toincreased gene expression.

Cells are grown for an extra 3 to 4 hours. Cells are then harvested bycentrifugation (20 mins at 600×g). The cell pellet is solubilized in thechaotropic agent 6 Molar Guanidine HCl by stirring for 3-4 hours at 4degree C. The cell debris is removed by centrifugation, and thesupernatant containing the polypeptide is loaded onto anickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column(available from QIAGEN, Inc., supra). Proteins with a 6×His tag bind tothe Ni-NTA resin with high affinity and can be purified in a simpleone-step procedure (for details see: The QIAexpressionist (1995) QIAGEN,Inc., supra).

Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl,pH 8, the column is first washed with 10 volumes of 6 M guanidine-HCl,pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finallythe polypeptide is eluted with 6 M guanidine-HCl, pH 5.

The purified protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni-NTA column. The recommended conditions areas follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl,20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins are eluted by the addition of 250 mMimidazole. Imidazole is removed by a final dialyzing step against PBS or50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified proteinis stored at 4 degree C. or frozen at −80 degree C.

Example 15 Purification of a Polypeptide from an Inclusion Body

The following alternative method can be used to purify a polypeptideexpressed in E coli when it is present in the form of inclusion bodies.Unless otherwise specified, all of the following steps are conducted at4-10 degree C.

Upon completion of the production phase of the E. coli fermentation, thecell culture is cooled to 4-10 degree C. and the cells harvested bycontinuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basisof the expected yield of protein per unit weight of cell paste and theamount of purified protein required, an appropriate amount of cellpaste, by weight, is suspended in a buffer solution containing 100 mMTris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneoussuspension using a high shear mixer.

The cells are then lysed by passing the solution through amicrofluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the polypeptidecontaining supernatant is incubated at 4 degree C. overnight to allowfurther GuHCl extraction.

Following high speed centrifugation (30,000×g) to remove insolubleparticles, the GuHCl solubilized protein is refolded by quickly mixingthe GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded dilutedprotein solution is kept at 4 degree C. without mixing for 12 hoursprior to further purification steps.

To clarify the refolded polypeptide solution, a previously preparedtangential filtration unit equipped with 0.16 um membrane filter withappropriate surface area (e.g., Filtron), equilibrated with 40 mM sodiumacetate, pH 6.0 is employed. The filtered sample is loaded onto a cationexchange resin (e.g., Poros HS-50, Perceptive Biosystems). The column iswashed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM,1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise manner. Theabsorbance at 280 nm of the effluent is continuously monitored.Fractions are collected and further analyzed by SDS-PAGE.

Fractions containing the polypeptide are then pooled and mixed with 4volumes of water. The diluted sample is then loaded onto a previouslyprepared set of tandem columns of strong anion (Poros HQ-50, PerceptiveBiosystems) and weak anion (Poros CM-20, Perceptive Biosystems) exchangeresins. The columns are equilibrated with 40 mM sodium acetate, pH 6.0.Both columns are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl.The CM-20 column is then eluted using a 10 column volume linear gradientranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCl, 50mM sodium acetate, pH 6.5. Fractions are collected under constant A280monitoring of the effluent. Fractions containing the polypeptide(determined, for instance, by 16% SDS-PAGE) are then pooled.

The resultant polypeptide should exhibit greater than 95% purity afterthe above refolding and purification steps. No major contaminant bandsshould be observed from Coomassie blue stained 16% SDS-PAGE gel when 5ug of purified protein is loaded. The purified protein can also betested for endotoxin/LPS contamination, and typically the LPS content isless than 0.1 ng/ml according to LAL assays.

Example 16 Cloning and Expression of a Polypeptide in a BaculovirusExpression System

In this example, the plasmid shuttle vector pAc373 is used to insert apolynucleotide into a baculovirus to express a polypeptide. A typicalbaculovirus expression vector contains the strong polyhedrin promoter ofthe Autographa californica nuclear polyhedrosis virus (AcMNPV) followedby convenient restriction sites, which may include, for example BamHI,Xba I and Asp718. The polyadenylation site of the simian virus 40(“SV40”) is often used for efficient polyadenylation. For easy selectionof recombinant virus, the plasmid contains the beta-galactosidase genefrom E. coli under control of a weak Drosophila promoter in the sameorientation, followed by the polyadenylation signal of the polyhedringene. The inserted genes are flanked on both sides by viral sequencesfor cell-mediated homologous recombination with wild-type viral DNA togenerate a viable virus that express the cloned polynucleotide.

Many other baculovirus vectors can be used in place of the vector above,such as pVL941 and pAcIMN1, as one skilled in the art would readilyappreciate, as long as the construct provides appropriately locatedsignals for transcription, translation, secretion and the like,including a signal peptide and an in-frame AUG as required. Such vectorsare described, for instance, in Luckow et al., Virology 170:31-39(1989).

A polynucleotide encoding a polypeptide of the present invention isamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ ends of the DNA sequence, as outlined in Example 13, to synthesizeinsertion fragments. The primers used to amplify the cDNA insert shouldpreferably contain restriction sites at the 5′ end of the primers inorder to clone the amplified product into the expression vector.Specifically, the cDNA sequence contained in the deposited clone,including the AUG initiation codon and the naturally associated leadersequence identified elsewhere herein (if applicable), is amplified usingthe PCR protocol described in Example 13. If the naturally occurringsignal sequence is used to produce the protein, the vector used does notneed a second signal peptide. Alternatively, the vector can be modifiedto include a baculovirus leader sequence, using the standard methodsdescribed in Summers et al., “A Manual of Methods for BaculovirusVectors and Insect Cell Culture Procedures,” Texas AgriculturalExperimental Station Bulletin No. 1555 (1987).

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with appropriate restrictionenzymes and again purified on a 1% agarose gel.

The plasmid is digested with the corresponding restriction enzymes andoptionally, can be dephosphorylated using calf intestinal phosphatase,using routine procedures known in the art. The DNA is then isolated froma 1% agarose gel using a commercially available kit (“Geneclean” BIO 101Inc., La Jolla, Calif.).

The fragment and the dephosphorylated plasmid are ligated together withT4 DNA ligase. E. coli HB 101 or other suitable E. coli hosts such asXL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells aretransformed with the ligation mixture and spread on culture plates.Bacteria containing the plasmid are identified by digesting DNA fromindividual colonies and analyzing the digestion product by gelelectrophoresis. The sequence of the cloned fragment is confirmed by DNAsequencing.

Five ug of a plasmid containing the polynucleotide is co-transformedwith 1.0 ug of a commercially available linearized baculovirus DNA(“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego, Calif.), usingthe lipofection method described by Felgner et al., Proc. Natl. Acad.Sci. USA 84:7413-7417 (1987). One ug of BaculoGoldtm virus DNA and 5 ugof the plasmid are mixed in a sterile well of a microtiter platecontaining 50 ul of serum-free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards, 10 ul Lipofectin plus 90 ul Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is then incubated for 5hours at 27 degrees C. The transfection solution is then removed fromthe plate and 1 ml of Grace's insect medium supplemented with 10% fetalcalf serum is added. Cultivation is then continued at 27 degrees C. forfour days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, supra. An agarose gel with“Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easyidentification and isolation of gal-expressing clones, which produceblue-stained plaques. (A detailed description of a “plaque assay” ofthis type can also be found in the user's guide for insect cell cultureand baculovirology distributed by Life Technologies Inc., Gaithersburg,page 9-10.) After appropriate incubation, blue stained plaques arepicked with the tip of a micropipettor (e.g., Eppendorf). The agarcontaining the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 ul of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4 degree C.

To verify the expression of the polypeptide, Sf9 cells are grown inGrace's medium supplemented with 10% heat-inactivated FBS. The cells areinfected with the recombinant baculovirus containing the polynucleotideat a multiplicity of infection (“MOI”) of about 2. If radiolabeledproteins are desired, 6 hours later the medium is removed and isreplaced with SF900 II medium minus methionine and cysteine (availablefrom Life Technologies Inc., Rockville, Md.). After 42 hours, 5 uCi of35S-methionine and 5 uCi 35S-cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then areharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins are analyzed by SDS-PAGE followed byautoradiography (if radiolabeled).

Microsequencing of the amino acid sequence of the amino terminus ofpurified protein may be used to determine the amino terminal sequence ofthe produced protein.

Example 17 Expression of a Polypeptide in Mammalian Cells

The polypeptide of the present invention can be expressed in a mammaliancell. A typical mammalian expression vector contains a promoter element,which mediates the initiation of transcription of mRNA, a protein codingsequence, and signals required for the termination of transcription andpolyadenylation of the transcript. Additional elements includeenhancers, Kozak sequences and intervening sequences flanked by donorand acceptor sites for RNA splicing. Highly efficient transcription isachieved with the early and late promoters from SV40, the long terminalrepeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the earlypromoter of the cytomegalovirus (CMV). However, cellular elements canalso be used (e.g., the human actin promoter).

Suitable expression vectors for use in practicing the present inventioninclude, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala,Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells thatcould be used include, human Hela, 293, H9 and Jurkat cells, mouseNIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse Lcells and Chinese hamster ovary (CHO) cells.

Alternatively, the polypeptide can be expressed in stable cell linescontaining the polynucleotide integrated into a chromosome. Theco-transformation with a selectable marker such as dhfr, gpt, neomycin,hygromycin allows the identification and isolation of the transformedcells.

The transformed gene can also be amplified to express large amounts ofthe encoded protein. The DHFR (dihydrofolate reductase) marker is usefulin developing cell lines that carry several hundred or even severalthousand copies of the gene of interest. (See, e.g., Alt, F. W., et al.,J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. and Ma, C., Biochem.et Biophys. Acta, 1097:107-143 (1990); Page, M. J. and Sydenham, M. A.,Biotechnology 9:64-68 (1991).) Another useful selection marker is theenzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279(1991); Bebbington et al., Bio/Technology 10:169-175 (1992). Using thesemarkers, the mammalian cells are grown in selective medium and the cellswith the highest resistance are selected. These cell lines contain theamplified gene(s) integrated into a chromosome. Chinese hamster ovary(CHO) and NSO cells are often used for the production of proteins.

A polynucleotide of the present invention is amplified according to theprotocol outlined in herein. If the naturally occurring signal sequenceis used to produce the protein, the vector does not need a second signalpeptide. Alternatively, if the naturally occurring signal sequence isnot used, the vector can be modified to include a heterologous signalsequence. (See, e.g., WO 96/34891.) The amplified fragment is isolatedfrom a 1% agarose gel using a commercially available kit (“Geneclean,”BIO 101 Inc., La Jolla, Calif.). The fragment then is digested withappropriate restriction enzymes and again purified on a 1% agarose gel.

The amplified fragment is then digested with the same restriction enzymeand purified on a 1% agarose gel. The isolated fragment and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC6 using,for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene is used fortransformation. Five pg of an expression plasmid is cotransformed with0.5 ug of the plasmid pSVneo using lipofectin (Felgner et al., supra).The plasmid pSV2-neo contains a dominant selectable marker, the neo genefrom Tn5 encoding an enzyme that confers resistance to a group ofantibiotics including G4 18. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of 100-200 uM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reversed phase HPLCanalysis.

Example 18 Protein Fusions

The polypeptides of the present invention are preferably fused to otherproteins. These fusion proteins can be used for a variety ofapplications. For example, fusion of the present polypeptides toHis-tag, HA-tag, protein A, IgG domains, and maltose binding proteinfacilitates purification. (See Example described herein; see also EP A394,827; Traunecker, et al., Nature 331:84-86 (1988).) Similarly, fusionto IgG-1, IgG-3, and albumin increases the half-life time in vivo.Nuclear localization signals fused to the polypeptides of the presentinvention can target the protein to a specific subcellular localization,while covalent heterodimer or homodimers can increase or decrease theactivity of a fusion protein. Fusion proteins can also create chimericmolecules having more than one function. Finally, fusion proteins canincrease solubility and/or stability of the fused protein compared tothe non-fused protein. All of the types of fusion proteins describedabove can be made by modifying the following protocol, which outlinesthe fusion of a polypeptide to an IgG molecule.

Briefly, the human Fc portion of the IgG molecule can be PCR amplified,using primers that span the 5′ and 3′ ends of the sequence describedbelow. These primers also should have convenient restriction enzymesites that will facilitate cloning into an expression vector, preferablya mammalian expression vector. Note that the polynucleotide is clonedwithout a stop codon, otherwise a fusion protein will not be produced.

The naturally occurring signal sequence may be used to produce theprotein (if applicable). Alternatively, if the naturally occurringsignal sequence is not used, the vector can be modified to include aheterologous signal sequence. (See, e.g., WO 96/34891 and/or U.S. Pat.No. 6,066,781, supra.)

Human IgG Fc region: (SEQ ID NO:48)GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 19 Method of Creating N- and C-terminal Deletion MutantsCorresponding to the CAN-12, CAN-12v1, and CAN-12v2 Polypeptides of thePresent Invention.

As described elsewhere herein, the present invention encompasses thecreation of N- and C-terminal deletion mutants, in addition to anycombination of N- and C-terminal deletions thereof, corresponding to theCAN-12, CAN-12v1, and CAN-12v2 polypeptides of the present invention. Anumber of methods are available to one skilled in the art for creatingsuch mutants. Such methods may include a combination of PCRamplification and gene cloning methodology. Although one of skill in theart of molecular biology, through the use of the teachings provided orreferenced herein, and/or otherwise known in the art as standardmethods, could readily create each deletion mutant of the presentinvention, exemplary methods are described below.

Briefly, using the isolated cDNA clone encoding the full-length CAN-12,CAN-12v1, or CAN-12v2 polypeptide sequence (as described in Example 13,for example), appropriate primers of about 15-25 nucleotides derivedfrom the desired 5′ and 3′ positions of SEQ ID NO: 1, 53, or 55 may bedesigned to PCR amplify, and subsequently clone, the intended N- and/orC-terminal deletion mutant. Such primers could comprise, for example, aninititation and stop codon for the 5′ and 3′ primer, respectively. Suchprimers may also comprise restriction sites to facilitate cloning of thedeletion mutant post amplification. Moreover, the primers may compriseadditional sequences, such as, for example, flag-tag sequences, kozacsequences, or other sequences discussed and/or referenced herein.

For example, in the case of the P23 to L581 CAN-12 N-terminal deletionmutant, the following primers could be used to amplify a cDNA fragmentcorresponding to this deletion mutant: 5′ Primer 5′-GCAGCA GCGGCCGCCCACAGCAACCCCAACAGGACTTTG-3′ (SEQ ID NO:49)             NotI 3′ Primer5′-GCAGCA GTCGAC TAACAAGGTGGTGTTGAAGATTAAA-3′ (SEQ ID NO:50)           SalI

For example, in the case of the M1 to L423 CAN-12 C-terminal deletionmutant, the following primers could be used to amplify a cDNA fragmentcorresponding to this deletion mutant: 5′ Primer 5′-GCAGCA GCGGCCGCATGTCTCTGTGGCCACCTTTCCG-3′ (SEQ ID NO:51)             NotI 3′ Primer5′-GCAGCA GTCGAC GAGGTAGAAGCCAATGGCGAGGAG-3′ (SEQ ID NO:52)           SalI

For example, in the case of the R90 to L694 CAN-12v1 N-terminal deletionmutant, the following primers could be used to amplify a cDNA fragmentcorresponding to this deletion mutant: 5′ Primer 5′-GCAGCA GCGGCCGCAGGCTGGATCTGTGCCAGGGGATAG-3′ (SEQ ID NO:94)              NotI 3′ Primer5′-GCAGCA GTCGAC TAACAAGGTGGTGTTGAAG-3′ (SEQ ID NO: 95)           SalI

For example, in the case of the M1 to G561 CAN-12v1 C-terminal deletionmutant, the following primers could be used to amplify a cDNA fragmentcorresponding to this deletion mutant: 5′ Primer 5′- GCAGCAGCGGCCGC ATGTCTCTGTGGCCACCTTTCCG -3′ (SEQ ID NO:96)              NotI3′ Primer 5′- GCAGCA GTCGAC CCCCTGGCAGGCTTCCAGGCTAAAG -3′ (SEQ ID NO:97)             SalI

For example, in the case of the R90 to L697 CAN-12v2 N-terminal deletionmutant, the following primers could be used to amplify a cDNA fragmentcorresponding to this deletion mutant: 5′ Primer 5′- GCAGCAGCGGCCGC AGGCTGGATCTGTGCCAGGGGATAG -3′ (SEQ ID NO:98)              NotI3′ Primer 5′- GCAGCA GTCGAC TAACAAGGTGGTGTTGAAG -3′         (SEQ IDNO:99)              SalI

For example, in the case of the MI to G564 CAN-12v2 C-terminal deletionmutant, the following primers could be used to amplify a cDNA fragmentcorresponding to this deletion mutant: 5′ Primer 5′- GCAGCAGCGGCCGC ATGTCTCTGTGGCCACCTTTCCG -3′ (SEQ ID NO:100)              NotI3′ Primer 5′- GCAGCA GTCGAC CCCCTGGCAGGCTTCCAGGCTAAAG -3′ (SEQ IDNO:101)              SalI

Representative PCR amplification conditions are provided below, althoughthe skilled artisan would appreciate that other conditions may berequired for efficient amplification. A 100 ul PCR reaction mixture maybe prepared using 10 ng of the template DNA (cDNA clone of CAN-12), 200uM 4dNTPs, 1 uM primers, 0.25U Taq DNA polymerase (PE), and standard TaqDNA polymerase buffer. Typical PCR cycling condition are as follows:20-25 cycles: 45 sec, 93 degrees  2 min, 50 degrees  2 min, 72 degrees  1 cycle: 10 min, 72 degrees

After the final extension step of PCR, 5U Klenow Fragment may be addedand incubated for 15 min at 30 degrees.

Upon digestion of the fragment with the NotI and SalI restrictionenzyres, the fragment could be cloned into an appropriate expressionand/or cloning vector which has been similarly digested (e.g., pSport1,among others). The skilled artisan would appreciate that other plasmidscould be equally substituted, and may be desirable in certaincircumstances. The digested fragment and vector are then ligated using aDNA ligase, and then used to transform competent E. coli cells usingmethods provided herein and/or otherwise known in the art.

The 5′ primer sequence for amplifying any additional N-terminal deletionmutants may be determined by reference to the following formula:

(S+(X*3)) to ((S+(X*3))+25), wherein ‘S’ is equal to the nucleotideposition of the initiating start codon of the CAN-12, CAN-12v1, orCAN12-v2 gene (SEQ ID NO: 1, 53, or 55), and ‘X’ is equal to the mostN-terminal amino acid of the intended N-terminal deletion mutant. Thefirst term will provide the start 5′ nucleotide position of the 5′primer, while the second term will provide the end 3′ nucleotideposition of the 5′ primer corresponding to sense strand of SEQ ID NO: 1,53, or 55. Once the corresponding nucleotide positions of the primer aredetermined, the final nucleotide sequence may be created by the additionof applicable restriction site sequences to the 5′ end of the sequence,for example. As referenced herein, the addition of other sequences tothe 5′ primer may be desired in certain circumstances (e.g., kozacsequences, etc.).

The 3′ primer sequence for amplifying any additional N-terminal deletionmutants may be determined by reference to the following formula:

(S+(X*3)) to ((S+(X*3))−25), wherein ‘S’ is equal to the nucleotideposition of the initiating start codon of the CAN-12, CAN-12v1, orCAN-12-v2 gene (SEQ ID NO:1, 53, or 55), and ‘X’ is equal to the mostC-terminal amino acid of the intended N-terminal deletion mutant. Thefirst term will provide the start 5′ nucleotide position of the 3′primer, while the second term will provide the end 3′ nucleotideposition of the 3′ primer corresponding to the anti-sense strand of SEQID NO: 1, 53, or 55. Once the corresponding nucleotide positions of theprimer are determined, the final nucleotide sequence may be created bythe addition of applicable restriction site sequences to the 5′ end ofthe sequence, for example. As referenced herein, the addition of othersequences to the 3′ primer may be desired in certain circumstances(e.g., stop codon sequences, etc.). The skilled artisan would appreciatethat modifications of the above nucleotide positions may be necessaryfor optimizing PCR amplification.

The same general formulas provided above may be used in identifying the5′ and 3′ primer sequences for amplifying any C-terminal deletion mutantof the present invention. Moreover, the same general formulas providedabove may be used in identifying the 5′ and 3′ primer sequences foramplifying any combination of N-terminal and C-terminal deletion mutantof the present invention. The skilled artisan would appreciate thatmodifications of the above nucleotide positions may be necessary foroptimizing PCR amplification.

Example 20 Site Directed/Site Specific Mutagenesis

In vitro site-directed mutagenesis is an invaluable technique forstudying protein structure-function relationships and gene expression,for example, as well as for vector modification. Site-directedmutagenesis can also be used for creating any of one or more of themutants of the present invention, particularly the conservative and/ornon-conservative amino acid substitution mutants of the presentinvention. Approaches utilizing single stranded DNA (ssDNA) as thetemplate have been reported (e.g., T. A. Kunkel et al., 1985, Proc.Natl. Acad. Sci. USA), 82:488-492; M. A. Vandeyar et al., 1988, Gene,65(1):129-133; M. Sugimoto et al., 1989, Anal. Biochem., 179(2):309-311; and J. W. Taylor et al., 1985, Nuc. Acids. Res., 13(24):8765-8785).

The use of PCR in site-directed mutagenesis accomplishes strandseparation by using a denaturing step to separate the complementarystrands and to allow efficient polymerization of the PCR primers. PCRsite-directed mutagenesis methods thus permit site specific mutations tobe incorporated in virtually any double stranded plasmid, thuseliminating the need for re-subcloning into M13-based bacteriophagevectors or single-stranded rescue. (M. P. Weiner et al., 1995, MolecularBiology: Current Innovations and Future Trends, Eds. A. M. Griffin andH. G. Griffin, Horizon Scientific Press, Norfolk, UK; and C. Papworth etal., 1996, Strategies, 9(3):3-4).

A protocol for performing site-directed mutagenesis, particularlyemploying the QuikChange™ site-directed mutagenesis kit (Stratagene, LaJolla, Calif.; U.S. Pat. Nos. 5,789,166 and 5,923,419) is provided formaking point mutations, to switch or substitute amino acids, and todelete or insert single or multiple amino acids in the RATL1d6 aminoacid sequence of this invention.

Primer Design

For primer design using this protocol, the mutagenic oligonucleotideprimers are designed individually according to the desired mutation. Thefollowing considerations should be made for designing mutagenicprimers: 1) Both of the mutagenic primers must contain the desiredmutation and anneal to the same sequence on opposite strands of theplasmid; 2) Primers should be between 25 and 45 bases in length, and themelting temperature (T_(m)) of the primers should be greater than, orequal to, 78° C. The following formula is commonly used for estimatingthe T_(m) of primers: T=81.5+0.41 (%GC)−675/N− %mismatch. Forcalculating T_(m), N is the primer length in bases; and values for %GCand % mismatch are whole numbers. For calculating T_(m) for primersintended to introduce insertions or deletions, a modified version of theabove formula is employed: T=81.5+0.41 (%GC)−675/N, where N does notinclude the bases which are being inserted or deleted; 3) The desiredmutation (deletion or insertion) should be in the middle of the primerwith approximately 10-15 bases of correct sequence on both sides; 4) Theprimers optimally should have a minimum GC content of 40%, and shouldterminate in one or more C or G bases; 5) Primers need not be5′-phosphorylated, but must be purified either by fast polynucleotideliquid chromatography (FPLC) or by polyacrylamide gel electrophoresis(PAGE). Failure to purify the primers results in a significant decreasein mutation efficiency; and 6). It is important that primerconcentration is in excess. It is suggested to vary the amount oftemplate while keeping the concentration of the primers constantly inexcess (QuikChange™ Site-Directed Mutagenesis Kit, Stratagene, La Jolla,Calif.).

Protocol for Setting UP the Reactions

Using the above-described primer design, two complimentaryoligonucleotides containing the desired mutation, flanked by unmodifiednucleic acid sequence, are synthesized. The resulting oligonucleotideprimers are purified.

A control reaction is prepared using 5 μl 10× reaction buffer (100 mMKCl; 100 mM (NH₄)₂SO₄; 200 mM Tris-HCl, pH 8.8; 20 mM MgSO₄; 1% Triton®X-100; 1 mg/ml nuclease-free bovine serum albumin, BSA); 2 μl (10 ng) ofpWhitescript™, 4.5-kb control plasmid (5 ng/μl); 1.25 μl (125 ng) ofoligonucleotide control primer #1 (34-mer, 100 ng/l); 1.25 μl (125 ng)of oligonucleotide control primer #2 (34-mer, 100 ng/μl); 1 μl of dNTPmix; double distilled H₂O; to a final volume of 50 μl. Thereafter, 1 μlof DNA polymerase (PfuTurbo® DNA Polymerase, Stratagene), (2.5 U/gl) isadded. PfuTurbo® DNA Polymerase is stated to have 6-fold higher fidelityin DNA synthesis than does Taq polymerase. To maximize temperaturecycling performance, use of thin-walled test tubes is suggested toensure optimum contact with the heating blocks of the temperaturecycler.

The sample reaction is prepared by combining 5 μl of 10× reactionbuffer; x μl (5-50 ng) of dsDNA template; x μl (125 ng) ofoligonucleotide primer #1; x μl (5-50 ng) of dsDNA template; x μg (125ng) of oligonucleotide primer #2; 1 μl of dNTP mix; and ddH₂O to a finalvolume of 50 μl. Thereafter, 1 μl of DNA polymerase (PfuTurbo DNAPolymerase, Stratagene), (2.5 U/gl) is added.

It is suggested that if the thermal cycler does not have a hot-topassembly, each reaction should be overlaid with approximately 30 μl ofmineral oil.

Cycling the Reactions

Each reaction is cycled using the following cycling parameters: SegmentCycles Temperature Time 1 1 95° C. 30 seconds 2 12-18 95° C. 30 seconds55° C. 1 minute 68° C. 2 minutes/kb of plasmid length

For the control reaction, a 12-minute extension time is used and thereaction is run for 12 cycles. Segment 2 of the above cycling parametersis adjusted in accordance with the type of mutation desired. Forexample, for point mutations, 12 cycles are used; for single amino acidchanges, 16 cycles are used; and for multiple amino acid deletions orinsertions, 18 cycles are used. Following the temperature cycling, thereaction is placed on ice for 2 minutes to cool the reaction to ≦37° C.

Digesting the Products and Transforming Competent Cells

One μl of the DpnI restriction enzyme (10 U/gl) is added directly (belowmineral oil overlay) to each amplification reaction using a small,pointed pipette tip. The reaction mixture is gently and thoroughly mixedby pipetting the solution up and down several times. The reactionmixture is then centrifuged for 1 minute in a microcentrifuge.Immediately thereafter, each reaction is incubated at 37° C. for 1 hourto digest the parental (i.e., the non-mutated) supercoiled dsDNA.

Competent cells (i.e., XL1-Blue supercompetent cells, Stratagene) arethawed gently on ice. For each control and sample reaction to betransformed, 50 μl of the supercompetent cells are aliquotted to aprechilled test tube (Falcon 2059 polypropylene). Next, 1 μl of theDpnI-digested DNA is transferred from the control and the samplereactions to separate aliquots of the supercompetent cells. Thetransformation reactions are gently swirled to mix and incubated for 30minutes on ice. Thereafter, the transformation reactions are heat-pulsedfor 45 seconds at 42° C. for 2 minutes.

0.5 ml of NZY+ broth, preheated to 42° C. is added to the transformationreactions which are then incubated at 37° C. for 1 hour with shaking at225-250 rpm. An aliquot of each transformation reaction is plated onagar plates containing the appropriate antibiotic for the vector. Forthe mutagenesis and transformation controls, cells are spread onLB-ampicillin agar plates containing 80 μg/ml of X-gal and 20 mM MIPTG.Transformation plates are incubated for >6 hours at 37° C.

Example 21 Regulation of Protein Expression Via Controlled Aggregationin the Endoplasmic Reticulum

As described more particularly herein, proteins regulate diversecellular processes in higher organisms, ranging from rapid metabolicchanges to growth and differentiation. Increased production of specificproteins could be used to prevent certain diseases and/or diseasestates. Thus, the ability to modulate the expression of specificproteins in an organism would provide significant benefits.

Numerous methods have been developed to date for introducing foreigngenes, either under the control of an inducible, constitutively active,or endogenous promoter, into organisms. Of particular interest are theinducible promoters (see, M. Gossen, et al., Proc. Natl. Acad. Sci.USA., 89:5547 (1992); Y. Wang, et al., Proc. Natl. Acad. Sci. USA,91:8180 (1994), D. No., et al., Proc. Natl. Acad. Sci. USA, 93:3346(1996); and V. M. Rivera, et al., Nature Med, 2:1028 (1996); in additionto additional examples disclosed elsewhere herein). In one example, thegene for erthropoietin (Epo) was transferred into mice and primatesunder the control of a small molecule inducer for expression (e.g.,tetracycline or rapamycin) (see, D. Bohl, et al., Blood, 92:1512,(1998); K. G. Rendahl, et al., Nat. Biotech, 16:757, (1998); V. M.Rivera, et al., Proc. Natl. Acad. Sci. USA, 96:8657 (1999); and X. Ye etal., Science, 283:88 (1999). Although such systems enable efficientinduction of the gene of interest in the organism upon addition of theinducing agent (i.e., tetracycline, rapamycin, etc,.), the levels ofexpression tend to peak at 24 hours and trail off to background levelsafter 4 to 14 days. Thus, controlled transient expression is virtuallyimpossible using these systems, though such control would be desirable.

A new alternative method of controlling gene expression levels of aprotein from a transgene (i.e., includes stable and transienttransformants) has recently been elucidated (V. M. Rivera., et al.,Science, 287:826-830, (2000)). This method does not control geneexpression at the level of the mRNA like the aforementioned systems.Rather, the system controls the level of protein in an active secretedform. In the absence of the inducing agent, the protein aggregates inthe ER and is not secreted. However, addition of the inducing agentresults in dis-aggregation of the protein and the subsequent secretionfrom the ER. Such a system affords low basal secretion, rapid, highlevel secretion in the presence of the inducing agent, and rapidcessation of secretion upon removal of the inducing agent. In fact,protein secretion reached a maximum level within 30 minutes ofinduction, and a rapid cessation of secretion within 1 hour of removingthe inducing agent. The method is also applicable for controlling thelevel of production for membrane proteins.

Detailed methods are presented in V. M. Rivera., et al., Science,287:826-830, (2000)), briefly:

Fusion protein constructs are created using polynucleotide sequences ofthe present invention with one or more copies (preferably at least 2, 3,4, or more) of a conditional aggregation domain (CAD) a domain thatinteracts with itself in a ligand-reversible manner (i.e., in thepresence of an inducing agent) using molecular biology methods known inthe art and discussed elsewhere herein. The CAD domain may be the mutantdomain isolated from the human FKBP12 (Phe³⁶ to Met) protein (asdisclosed in V. M. Rivera., et al., Science, 287:826-830, (2000), oralternatively other proteins having domains with similarligand-reversible, self-aggregation properties. As a principle of designthe fusion protein vector would contain a furin cleavage sequenceoperably linked between the polynucleotides of the present invention andthe CAD domains. Such a cleavage site would enable the proteolyticcleavage of the CAD domains from the polypeptide of the presentinvention subsequent to secretion from the ER and upon entry into thetrans-Golgi (J. B. Denault, et al., FEBS Lett., 379:113, (1996)).Alternatively, the skilled artisan would recognize that any proteolyticcleavage sequence could be substituted for the furin sequence providedthe substituted sequence is cleavable either endogenously (e.g., thefurin sequence) or exogenously (e.g., post secretion, post purification,post production, etc.). The preferred sequence of each feature of thefusion protein construct, from the 5′ to 3′ direction with each featurebeing operably linked to the other, would be a promoter, signalsequence, “X” number of (CAD)x domains, the furin sequence (or otherproteolytic sequence), and the coding sequence of the polypeptide of thepresent invention. The artisan would appreciate that the promotor andsignal sequence, independent from the other, could be either theendogenous promotor or signal sequence of a polypeptide of the presentinvention, or alternatively, could be a heterologous signal sequence andpromotor.

The specific methods described herein for controlling protein secretionlevels through controlled ER aggregation are not meant to be limitingare would be generally applicable to any of the polynucleotides andpolypeptides of the present invention, including variants, homologues,orthologs, and fragments therein.

Example 22 Alteration of Protein Glycosylation Sites to EnhanceCharacteristics of Polypeptides of the Invention

Many eukaryotic cell surface and proteins are post-translationallyprocessed to incorporate N-linked and O-linked carbohydrates (Kornfeldand Kornfeld (1985) Annu. Rev. Biochem. 54:631-64; Rademacher et al.,(1988) Annu. Rev. Biochem. 57:785-838). Protein glycosylation is thoughtto serve a variety of functions including: augmentation of proteinfolding, inhibition of protein aggregation, regulation of intracellulartrafficking to organelles, increasing resistance to proteolysis,modulation of protein antigenicity, and mediation of intercellularadhesion (Fieldler and Simons (1995) Cell, 81:309-312; Helenius (1994)Mol. Biol. Of the Cell 5:253-265; Olden et al., (1978) Cell, 13:461-473;Caton et al., (1982) Cell, 37:417-427; Alexander and Elder (1984),Science, 226:1328-1330; and Flack et al., (1994), J. Biol. Chem.,269:14015-14020). In higher organisms, the nature and extent ofglycosylation can markedly affect the circulating half-life andbio-availability of proteins by mechanisms involving receptor mediateduptake and clearance (Ashwell and Morrell, (1974), Adv. Enzymol.,41:99-128; Ashwell and Harford (1982), Ann. Rev. Biochem., 51:531-54).Receptor systems have been identified that are thought to play a majorrole in the clearance of serum proteins through recognition of variouscarbohydrate structures on the glycoproteins (Stockert (1995), Physiol.Rev., 75:591-609; Kery et al., (1992), Arch. Biochem. Biophys.,298:49-55). Thus, production strategies resulting in incompleteattachment of terminal sialic acid residues might provide a means ofshortening the bioavailability and half-life of glycoproteins.Conversely, expression strategies resulting in saturation of terminalsialic acid attachment sites might lengthen protein bioavailability andhalf-life.

In the development of recombinant glycoproteins for use aspharmaceutical products, for example, it has been speculated that thepharmacodynamics of recombinant proteins can be modulated by theaddition or deletion of glycosylation sites from a glycoproteins primarystructure (Berman and Lasky (1985a) Trends in Biotechnol., 3:51-53).However, studies have reported that the deletion of N-linkedglycosylation sites often impairs intracellular transport and results inthe intracellular accumulation of glycosylation site variants (Machamerand Rose (1988), J. Biol Chem., 263:5955-5960; Gallagher et al., (1992),J. Virology., 66:7136-7145; Collier et al., (1993), Biochem.,32:7818-7823; Claffey et al., (1995) Biochemica et Biophysica Acta,1246:1-9; Dube et al., (1988), J. Biol. Chem. 263:17516-17521). Whileglycosylation site variants of proteins can be expressedintracellularly, it has proved difficult to recover useful quantitiesfrom growth conditioned cell culture medium.

Moreover, it is unclear to what extent a glycosylation site in onespecies will be recognized by another species glycosylation machinery.Due to the importance of glycosylation in protein metabolism,particularly the secretion and/or expression of the protein, whether aglycosylation signal is recognized may profoundly determine a proteinsability to be expressed, either endogenously or recombinately, inanother organism (i.e., expressing a human protein in E. coli, yeast, orviral organisms; or an E. coli, yeast, or viral protein in human, etc.).Thus, it may be desirable to add, delete, or modify a glycosylationsite, and possibly add a glycosylation site of one species to a proteinof another species to improve the proteins functional, bioprocesspurification, and/or structural characteristics (e.g., a polypeptide ofthe present invention).

A number of methods may be employed to identify the location ofglycosylation sites within a protein. One preferred method is to run thetranslated protein sequence through the PROSITE computer program (SwissInstitute of Bioinformatics). Once identified, the sites could besystematically deleted, or impaired, at the level of the DNA usingmutagenesis methodology known in the art and available to the skilledartisan, Preferably using PCR-directed mutagenesis (See Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring, N.Y. (1982)). Similarly, glycosylation sites could be added, ormodified at the level of the DNA using similar methods, preferably PCRmethods (See, Maniatis, supra). The results of modifying theglycosylation sites for a particular protein (e.g., solubility,secretion potential, activity, aggregation, proteolytic resistance,etc.) could then be analyzed using methods know in the art.

The skilled artisan would acknowledge the existence of other computeralgorithms capable of predicting the location of glycosylation siteswithin a protein. For example, the Motif computer program (GeneticsComputer Group suite of programs) provides this function, as well.

Example 23 Method of Enhancing the Biological Activity/FunctionalCharacteristics of Invention through Molecular Evolution

Although many of the most biologically active proteins known are highlyeffective for their specified function in an organism, they oftenpossess characteristics that make them undesirable for transgenic,therapeutic, and/or industrial applications. Among these traits, a shortphysiological half-life is the most prominent problem, and is presenteither at the level of the protein, or the level of the proteins mRNA.The ability to extend the half-life, for example, would be particularlyimportant for a proteins use in gene therapy, transgenic animalproduction, the bioprocess production and purification of the protein,and use of the protein as a chemical modulator among others. Therefore,there is a need to identify novel variants of isolated proteinspossessing characteristics which enhance their application as atherapeutic for treating diseases of animal origin, in addition to theproteins applicability to common industrial and pharmaceuticalapplications.

Thus, one aspect of the present invention relates to the ability toenhance specific characteristics of invention through directed molecularevolution. Such an enhancement may, in a non-limiting example, benefitthe inventions utility as an essential component in a kit, theinventions physical attributes such as its solubility, structure, orcodon optimization, the inventions specific biological activity,including any associated enzymatic activity, the proteins enzymekinetics, the proteins Ki, Kcat, Km, Vmax, Kd, protein-protein activity,protein-DNA binding activity, antagonist/inhibitory activity (includingdirect or indirect interaction), agonist activity (including direct orindirect interaction), the proteins antigenicity (e.g., where it wouldbe desirable to either increase or decrease the antigenic potential ofthe protein), the immunogenicity of the protein, the ability of theprotein to form dimers, trimers, or multimers with either itself orother proteins, the antigenic efficacy of the invention, including itssubsequent use a preventative treatment for disease or disease states,or as an effector for targeting diseased genes. Moreover, the ability toenhance specific characteristics of a protein may also be applicable tochanging the characterized activity of an enzyme to an activitycompletely unrelated to its initially characterized activity. Otherdesirable enhancements of the invention would be specific to eachindividual protein, and would thus be well known in the art andcontemplated by the present invention.

For example, an engineered calpain may be constitutively active uponbinding of its cognate substrate. Alternatively, an engineered calpainmay be constitutively active in the absence of substrate binding, and/ormay exhibit increased efficacy in inhibiting cysteine proteases. In yetanother example, an engineered calpain may be capable of being activatedwith less than all of the regulatory factors and/or conditions typicallyrequired for calpain activation (e.g., substrate binding,phosphorylation, cofactor binding, Ca+ binding, Ca+ activation,conformational changes, etc.). Such calpain would be useful in screensto identify calpain modulators, among other uses described herein.

Directed evolution is comprised of several steps. The first step is toestablish a library of variants for the gene or protein of interest. Themost important step is to then select for those variants that entail theactivity you wish to identify. The design of the screen is essentialsince your screen should be selective enough to eliminate non-usefulvariants, but not so stringent as to eliminate all variants. The laststep is then to repeat the above steps using the best variant from theprevious screen. Each successive cycle, can then be tailored asnecessary, such as increasing the stringency of the screen, for example.

Over the years, there have been a number of methods developed tointroduce mutations into macromolecules. Some of these methods include,random mutagenesis, “error-prone” PCR, chemical mutagenesis,site-directed mutagenesis, and other methods well known in the art (fora comprehensive listing of current mutagenesis methods, see Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring, N.Y. (1982)). Typically, such methods have been used, forexample, as tools for identifying the core functional region(s) of aprotein or the function of specific domains of a protein (if amulti-domain protein). However, such methods have more recently beenapplied to the identification of macromolecule variants with specific orenhanced characteristics.

Random mutagenesis has been the most widely recognized method to date.Typically, this has been carried out either through the use of“error-prone” PCR (as described in Moore, J., et al, NatureBiotechnology 14:458, (1996), or through the application of randomizedsynthetic oligonucleotides corresponding to specific regions of interest(as described by Derbyshire, K. M. et al, Gene, 46:145-152, (1986), andHill, D E, et al, Methods Enzymol., 55:559-568, (1987). Both approacheshave limits to the level of mutagenesis that can be obtained. However,either approach enables the investigator to effectively control the rateof mutagenesis. This is particularly important considering the fact thatmutations beneficial to the activity of the enzyme are fairly rare. Infact, using too high a level of mutagenesis may counter or inhibit thedesired benefit of a useful mutation.

While both of the aforementioned methods are effective for creatingrandomized pools of macromolecule variants, a third method, termed “DNAShuffling”, or “sexual PCR” (WPC, Stemmer, PNAS, 91:10747, (1994)) hasrecently been elucidated. DNA shuffling has also been referred to as“directed molecular evolution”, “exon-shuffling”, “directed enzymeevolution”, in vitro evolution”, and “artificial evolution”. Suchreference terms are known in the art and are encompassed by theinvention. This new, preferred, method apparently overcomes thelimitations of the previous methods in that it not only propagatespositive traits, but simultaneously eliminates negative traits in theresulting progeny.

DNA shuffling accomplishes this task by combining the principal of invitro recombination, along with the method of “error-prone” PCR. Ineffect, you begin with a randomly digested pool of small fragments ofyour gene, created by Dnase I digestion, and then introduce said randomfragments into an “error-prone” PCR assembly reaction. During the PCRreaction, the randomly sized DNA fragments not only hybridize to theircognate strand, but also may hybridize to other DNA fragmentscorresponding to different regions of the polynucleotide ofinterest—regions not typically accessible via hybridization of theentire polynucleotide. Moreover, since the PCR assembly reactionutilizes “error-prone” PCR reaction conditions, random mutations areintroduced during the DNA synthesis step of the PCR reaction for all ofthe fragments -further diversifying the potential hybridization sitesduring the annealing step of the reaction.

A variety of reaction conditions could be utilized to carry-out the DNAshuffling reaction. However, specific reaction conditions for DNAshuffling are provided, for example, in PNAS, 91:10747, (1994). Briefly:

Prepare the DNA substrate to be subjected to the DNA shuffling reaction.Preparation may be in the form of simply purifying the DNA fromcontaminating cellular material, chemicals, buffers, oligonucleotideprimers, deoxynucleotides, RNAs, etc., and may entail the use of DNApurification kits as those provided by Qiagen, Inc., or by the Promega,Corp., for example.

Once the DNA substrate has been purified, it would be subjected to DnaseI digestion. About 2-4 ug of the DNA substrate(s) would be digested with0.0015 units of Dnase I (Sigma) per ul in 100 ul of 50 mM Tris-HCl, pH7.4/1 mM MgCl2 for 10-20 min. at room temperature. The resultingfragments of 10-50 bp could then be purified by running them through a2% low-melting point agarose gel by electrophoresis onto DE81ion-exchange paper (Whatmann) or could be purified using Microconconcentrators (Amicon) of the appropriate molecular weight cutoff, orcould use oligonucleotide purification columns (Qiagen), in addition toother methods known in the art. If using DE81 ion-exchange paper, the10-50 bp fragments could be eluted from said paper using 1M NaCl,followed by ethanol precipitation.

The resulting purified fragments would then be subjected to a PCRassembly reaction by re-suspension in a PCR mixture containing: 2 mM ofeach dNTP, 2.2 mM MgCl2, 50 mM KCl, 10 mM TriseHCl, pH 9.0, and 0.1%Triton X-100, at a final fragment concentration of 10-30 ng/ul. Noprimers are added at this point. Taq DNA polymerase (Promega) would beused at 2.5 units per 100 ul of reaction mixture. A PCR program of 94 Cfor 60s; 94 C for 30s, 50-55 C for 30s, and 72 C for 30s using 3045cycles, followed by 72 C for 5 min using an MJ Research (Cambridge,Mass.) PTC-150 thermocycler. After the assembly reaction is completed, a1:40 dilution of the resulting primerless product would then beintroduced into a PCR mixture (using the same buffer mixture used forthe assembly reaction) containing 0.8 um of each primer and subjectingthis mixture to 15 cycles of PCR (using 94 C for 30s, 5° C. for 30s, and72 C for 30s). The referred primers would be primers corresponding tothe nucleic acid sequences of the polynucleotide(s) utilized in theshuffling reaction. Said primers could consist of modified nucleic acidbase pairs using methods known in the art and referred to else whereherein, or could contain additional sequences (i.e., for addingrestriction sites, mutating specific base-pairs, etc.).

The resulting shuffled, assembled, and amplified product can be purifiedusing methods well known in the art (e.g., Qiagen PCR purification kits)and then subsequently cloned using appropriate restriction enzymes.

Although a number of variations of DNA shuffling have been published todate, such variations would be obvious to the skilled artisan and areencompassed by the invention. The DNA shuffling method can also betailored to the desired level of mutagenesis using the methods describedby Zhao, et al. (Nucl Acid Res., 25(6):1307-1308, (1997).

As described above, once the randomized pool has been created, it canthen be subjected to a specific screen to identify the variantpossessing the desired characteristic(s). Once the variant has beenidentified, DNA corresponding to the variant could then be used as theDNA substrate for initiating another round of DNA shuffling. This cycleof shuffling, selecting the optimized variant of interest, and thenre-shuffling, can be repeated until the ultimate variant is obtained.Examples of model screens applied to identify variants created using DNAshuffling technology may be found in the following publications: J. C.,Moore, et al., J. Mol. Biol., 272:336-347, (1997), F. R., Cross, et al.,Mol. Cell. Biol., 18:2923-2931, (1998), and A. Crameri., et al., Nat.Biotech., 15:436-438, (1997).

DNA shuffling has several advantages. First, it makes use of beneficialmutations. When combined with screening, DNA shuffling allows thediscovery of the best mutational combinations and does not assume thatthe best combination contains all the mutations in a population.Secondly, recombination occurs simultaneously with point mutagenesis. Aneffect of forcing DNA polymerase to synthesize full-length genes fromthe small fragment DNA pool is a background mutagenesis rate. Incombination with a stringent selection method, enzymatic activity hasbeen evolved up to 16000 fold increase over the wild-type form of theenzyme. In essence, the background mutagenesis yielded the geneticvariability on which recombination acted to enhance the activity.

A third feature of recombination is that it can be used to removedeleterious mutations. As discussed above, during the process of therandomization, for every one beneficial mutation, there may be at leastone or more neutral or inhibitory mutations. Such mutations can beremoved by including in the assembly reaction an excess of the wild-typerandom-size fragments, in addition to the random-size fragments of theselected mutant from the previous selection. During the next selection,some of the most active variants of thepolynucleotide/polypeptide/enzyme, should have lost the inhibitorymutations.

Finally, recombination enables parallel processing. This represents asignificant advantage since there are likely multiple characteristicsthat would make a protein more desirable (e.g. solubility, activity,etc.). Since it is increasingly difficult to screen for more than onedesirable trait at a time, other methods of molecular evolution tend tobe inhibitory. However, using recombination, it would be possible tocombine the randomized fragments of the best representative variants forthe various traits, and then select for multiple properties at once.

DNA shuffling can also be applied to the polynucleotides andpolypeptides of the present invention to decrease their immunogenicityin a specified host. For example, a particular variant of the presentinvention may be created and isolated using DNA shuffling technology.Such a variant may have all of the desired characteristics, though maybe highly immunogenic in a host due to its novel intrinsic structure.Specifically, the desired characteristic may cause the polypeptide tohave a non-native structure which could no longer be recognized as a“self” molecule, but rather as a “foreign”, and thus activate a hostimmune response directed against the novel variant. Such a limitationcan be overcome, for example, by including a copy of the gene sequencefor a xenobiotic ortholog of the native protein in with the genesequence of the novel variant gene in one or more cycles of DNAshuffling. The molar ratio of the ortholog and novel variant DNAs couldbe varied accordingly. Ideally, the resulting hybrid variant identifiedwould contain at least some of the coding sequence which enabled thexenobiotic protein to evade the host immune system, and additionally,the coding sequence of the original novel variant that provided thedesired characteristics.

Likewise, the invention encompasses the application of DNA shufflingtechnology to the evolution of polynucleotides and polypeptides of theinvention, wherein one or more cycles of DNA shuffling include, inaddition to the gene template DNA, oligonucleotides coding for knownallelic sequences, optimized codon sequences, known variant sequences,known polynucleotide polymorphism sequences, known ortholog sequences,known homologue sequences, additional homologous sequences, additionalnon-homologous sequences, sequences from another species, and any numberand combination of the above.

In addition to the described methods above, there are a number ofrelated methods that may also be applicable, or desirable in certaincases. Representative among these are the methods discussed in PCTapplications WO 98/31700, and WO 98/32845, which are hereby incorporatedby reference. Furthermore, related methods can also be applied to thepolynucleotide sequences of the present invention in order to evolveinvention for creating ideal variants for use in gene therapy, proteinengineering, evolution of whole cells containing the variant, or in theevolution of entire enzyme pathways containing polynucleotides of theinvention as described in PCT applications WO 98/13485, WO 98/13487, WO98/27230, WO 98/31837, and Crameri, A., et al., Nat. Biotech.,15:436-438, (1997), respectively.

Additional methods of applying “DNA Shuffling” technology to thepolynucleotides and polypeptides of the present invention, includingtheir proposed applications, may be found in U.S. Pat. No. 5,605,793;PCT Application No. WO 95/22625; PCT Application No. WO 97/20078; PCTApplication No. WO 97/35966; and PCT Application No. WO 98/42832; PCTApplication No. WO 00/09727 specifically provides methods for applyingDNA shuffling to the identification of herbicide selective crops whichcould be applied to the polynucleotides and polypeptides of the presentinvention; additionally, PCT Application No. WO 00/12680 providesmethods and compositions for generating, modifying, adapting, andoptimizing polynucleotide sequences that confer detectable phenotypicproperties on plant species; each of the above are hereby incorporatedin their entirety herein for all purposes.

Example 24 Method of Determining Alterations in a Gene Corresponding toa Polynucleotide

RNA isolated from entire families or individual patients presenting witha phenotype of interest (such as a disease) is be isolated. cDNA is thengenerated from these RNA samples using protocols known in the art. (See,Sambrook.) The cDNA is then used as a template for PCR, employingprimers surrounding regions of interest in SEQ ID NO:1. Suggested PCRconditions consist of 35 cycles at 95 degrees C. for 30 seconds; 60-120seconds at 52-58 degrees C.; and 60-120 seconds at 70 degrees C., usingbuffer solutions described in Sidransky et al., Science 252:706 (1991).

PCR products are then sequenced using primers labeled at their 5′ endwith T4 polynucleotide kinase, employing SequiTherm Polymerase.(Epicentre Technologies). The intron-exon borders of selected exons isalso determined and genomic PCR products analyzed to confirm theresults. PCR products harboring suspected mutations is then cloned andsequenced to validate the results of the direct sequencing.

PCR products is cloned into T-tailed vectors as described in Holton etal., Nucleic Acids Research, 19:1156 (1991) and sequenced with T7polymerase (United States Biochemical). Affected individuals areidentified by mutations not present in unaffected individuals.

Genomic rearrangements are also observed as a method of determiningalterations in a gene corresponding to a polynucleotide. Genomic clonesisolated according to Example 2 are nick-translated withdigoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), and FISHperformed as described in Johnson et al., Methods Cell Biol. 35:73-99(1991). Hybridization with the labeled probe is carried out using a vastexcess of human cot-i DNA for specific hybridization to thecorresponding genomic locus.

Chromosomes are counterstained with 4,6-diamino-2-phenylidole andpropidium iodide, producing a combination of C- and R-bands. Alignedimages for precise mapping are obtained using a triple-band filter set(Chroma Technology, Brattleboro, Vt.) in combination with a cooledcharge-coupled device camera (Photometrics, Tucson, Ariz.) and variableexcitation wavelength filters. (Johnson et al., Genet. Anal. Tech.Appl., 8:75 (1991).) Image collection, analysis and chromosomalfractional length measurements are performed using the ISee GraphicalProgram System. (Inovision Corporation, Durham, N.C.) Chromosomealterations of the genomic region hybridized by the probe are identifiedas insertions, deletions, and translocations. These alterations are usedas a diagnostic marker for an associated disease.

Example 25 Method of Detecting Abnormal Levels of a Polypeptide in aBiological Sample

A polypeptide of the present invention can be detected in a biologicalsample, and if an increased or decreased level of the polypeptide isdetected, this polypeptide is a marker for a particular phenotype.Methods of detection are numerous, and thus, it is understood that oneskilled in the art can modify the following assay to fit theirparticular needs.

For example, antibody-sandwich ELISAs are used to detect polypeptides ina sample, preferably a biological sample. Wells of a microtiter plateare coated with specific antibodies, at a final concentration of 0.2 to10 ug/ml. The antibodies are either monoclonal or polyclonal and areproduced by the method described elsewhere herein. The wells are blockedso that non-specific binding of the polypeptide to the well is reduced.

The coated wells are then incubated for >2 hours at RT with a samplecontaining the polypeptide. Preferably, serial dilutions of the sampleshould be used to validate results. The plates are then washed threetimes with deionized or distilled water to remove unbounded polypeptide.

Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at aconcentration of 25-400 ng, is added and incubated for 2 hours at roomtemperature. The plates are again washed three times with deionized ordistilled water to remove unbounded conjugate.

Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenylphosphate (NPP) substrate solution to each well and incubate I hour atroom temperature. Measure the reaction by a microtiter plate reader.Prepare a standard curve, using serial dilutions of a control sample,and plot polypeptide concentration on the X-axis (log.scale) andfluorescence or absorbance of the Y-axis (linear scale). Interpolate theconcentration of the polypeptide in the sample using the standard curve.

Example 26 Formulation

The invention also provides methods of treatment and/or preventiondiseases, disorders, and/or conditions (such as, for example, any one ormore of the diseases or disorders disclosed herein) by administration toa subject of an effective amount of a Therapeutic. By therapeutic ismeant a polynucleotides or polypeptides of the invention (includingfragments and variants), agonists or antagonists thereof, and/orantibodies thereto, in combination with a pharmaceutically acceptablecarrier type (e.g., a sterile carrier).

The Therapeutic will be formulated and dosed in a fashion consistentwith good medical practice, taking into account the clinical conditionof the individual patient (especially the side effects of treatment withthe Therapeutic alone), the site of delivery, the method ofadministration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” for purposes herein isthus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount ofthe Therapeutic administered parenterally per dose will be in the rangeof about 1 ug/kg/day to 10 mg/kg/day of patient body weight, although,as noted above, this will be subject to therapeutic discretion. Morepreferably, this dose is at least 0.01 mg/kg/day, and most preferablyfor humans between about 0.01 and 1 mg/kg/day for the hormone. If givencontinuously, the Therapeutic is typically administered at a dose rateof about 1 ug/kg/hour to about 50 ug/kg/hour, either by 14 injectionsper day or by continuous subcutaneous infusions, for example, using amini-pump. An intravenous bag solution may also be employed. The lengthof treatment needed to observe changes and the interval followingtreatment for responses to occur appears to vary depending on thedesired effect.

Therapeutics can be administered orally, rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any. The term “parenteral” as usedherein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

In yet an additional embodiment, the Therapeutics of the invention aredelivered orally using the drug delivery technology described in U.S.Pat. No. 6,258,789, which is hereby incorporated by reference herein.

Therapeutics of the invention are also suitably administered bysustained-release systems. Suitable examples of sustained-releaseTherapeutics are administered orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral” asused herein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

Therapeutics of the invention may also be suitably administered bysustained-release systems. Suitable examples of sustained-releaseTherapeutics include suitable polymeric materials (such as, for example,semi-permeable polymer matrices in the form of shaped articles, e.g.,films, or microcapsules), suitable hydrophobic materials (for example asan emulsion in an acceptable oil) or ion exchange resins, and sparinglysoluble derivatives (such as, for example, a sparingly soluble salt).

Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)),poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater.Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)),ethylene vinyl acetate (Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

Sustained-release Therapeutics also include liposomally entrappedTherapeutics of the invention (see, generally, Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,N.Y., pp. 317 -327 and 353-365 (1989)). Liposomes containing theTherapeutic are prepared by methods known per se: DE 3,218,121; Epsteinet al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci.(USA) 77:4030-4034 (1980); EP 52,322; EP 36,676;EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S.Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, theliposomes are of the small (about 200-800 Angstroms) unilamellar type inwhich the lipid content is greater than about 30 mol. percentcholesterol, the selected proportion being adjusted for the optimalTherapeutic.

In yet an additional embodiment, the Therapeutics of the invention aredelivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref.Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);Saudek et al., N. Engl. J. Med. 321:574 (1989)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

For parenteral administration, in one embodiment, the Therapeutic isformulated generally by mixing it at the desired degree of purity, in aunit dosage injectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to the Therapeutic.

Generally, the formulations are prepared by contacting the Therapeuticuniformly and intimately with liquid carriers or finely divided solidcarriers or both. Then, if necessary, the product is shaped into thedesired formulation. Preferably the carrier is a parenteral carrier,more preferably a solution that is isotonic with the blood of therecipient. Examples of such carrier vehicles include water, saline,Ringer's solution, and dextrose solution. Non-aqueous vehicles such asfixed oils and ethyl oleate are also useful herein, as well asliposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

The Therapeutic will typically be formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, ata pH of about 3 to 8. It will be understood that the use of certain ofthe foregoing excipients, carriers, or stabilizers will result in theformation of polypeptide salts.

Any pharmaceutical used for therapeutic administration can be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeuticsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

Therapeutics ordinarily will be stored in unit or multi-dose containers,for example, sealed ampoules or vials, as an aqueous solution or as alyophilized formulation for reconstitution. As an example of alyophilized formulation, 10-ml vials are filled with 5 ml ofsterile-filtered 1% (w/v) aqueous Therapeutic solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized Therapeutic using bacteriostaticWater-for-Injection.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of theTherapeutics of the invention. Associated with such container(s) can bea notice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration. In addition, the Therapeutics may be employedin conjunction with other therapeutic compounds.

The Therapeutics of the invention may be administered alone or incombination with adjuvants. Adjuvants that may be administered with theTherapeutics of the invention include, but are not limited to, alum,alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21(Genentech, Inc.), BCG, and MPL. In a specific embodiment, Therapeuticsof the invention are administered in combination with alum. In anotherspecific embodiment, Therapeutics of the invention are administered incombination with QS-21. Further adjuvants that may be administered withthe Therapeutics of the invention include, but are not limited to,Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18,CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology.Vaccines that may be administered with the Therapeutics of the inventioninclude, but are not limited to, vaccines directed toward protectionagainst MMR (measles, mumps, rubella), polio, varicella,tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B,whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus,cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies,typhoid fever, and pertussis. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

The Therapeutics of the invention may be administered alone or incombination with other therapeutic agents. Therapeutic agents that maybe administered in combination with the Therapeutics of the invention,include but not limited to, other members of the TNF family,chemotherapeutic agents, antibiotics, steroidal and non-steroidalanti-inflammatories, conventional immunotherapeutic agents, cytokinesand/or growth factors. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

In one embodiment, the Therapeutics of the invention are administered incombination with members of the TNF family. TNF, TNF-related or TNF-likemolecules that may be administered with the Therapeutics of theinvention include, but are not limited to, soluble forms of TNF-alpha,lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found incomplex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L,4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO96/14328), AIM-I (International Publication No. WO 97/33899),endokine-alpha (International Publication No. WO 98/07880), TR6(International Publication No. WO 98/30694), OPG, and neutrokine-alpha(International Publication No. WO 98/18921, OX40, and nerve growthfactor (NOF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2(International Publication No. WO 96/34095), DR3 (InternationalPublication No. WO 97/33904), DR4 (International Publication No. WO98/32856), TR5 (International Publication No. WO 98/30693), TR6(International Publication No. WO 98/30694), TR7 (InternationalPublication No. WO 98/41629), TRANK, TR9 (International Publication No.WO 98/56892),TR10 (International Publication No. WO 98/54202), 312C2(International Publication No. WO 98/06842), and TR12, and soluble formsCD154, CD70, and CD153.

In certain embodiments, Therapeutics of the invention are administeredin combination with antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors. Nucleoside reverse transcriptaseinhibitors that may be administered in combination with the Therapeuticsof the invention, include, but are not limited to, RETROVIR(zidovudine/AZT), VIDEX (didanosine/ddI), HIVID (zalcitabine/ddC), ZERIT(stavudine/d4T), EPIVIR (lamivudine/3TC), and COMBIVIR(zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitorsthat may be administered in combination with the Therapeutics of theinvention, include, but are not limited to, VIRAMUE (nevirapine),RESCRIPTOR (delavirdine), and SUSTIVA (efavirenz). Protease inhibitorsthat may be administered in combination with the Therapeutics of theinvention, include, but are not limited to, CRIXIVAN (indinavir), NORVIR(ritonavir), INVIRASE (saquinavir), and VIRACEPT (nelfinavir). In aspecific embodiment, antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors may be used in any combinationwith Therapeutics of the invention to treat AIDS and/or to prevent ortreat HIV infection.

In other embodiments, Therapeutics of the invention may be administeredin combination with anti-opportunistic infection agents.Anti-opportunistic agents that may be administered in combination withthe Therapeutics of the invention, include, but are not limited to,TRIMETHOPRIM-SULFAMETHOXAZOLE, DAPSONE, PENTAMIDINE, ATOVAQUONE,ISONIAZID, RIFAMPIN, PYRAZIAMIDE, ETHAMBUTOL, RIFABUTIN, CLARITHROMYCIN,AZITHROMYCIN, GANCICLOVK, FOSCARNET, CIDOFOVK, FLUCONAZOLE,ITRACONAZOLE, KETOCONAZOLE, ACYCLOVIR, FAMCICOLVIR, PYRIMETHAMMNE,LEUCOVORIN, NEUPOGEN (filgrastim/G-CSF), and LEUKINE(sargramostim/GM-CSF). In a specific embodiment, Therapeutics of theinvention are used in any combination withTRIMETHOPRIM-SULFAMETHOXAZOLE, DAPSONE, PENTAMIDINE, and/or ATOVAQUONEto prophylactically treat or prevent an opportunistic Pneumocystiscarinii pneumonia infection. In another specific embodiment,Therapeutics of the invention are used in any combination withISONLAZID, RIFAMPIN, PYRAZINAMIDE, and/or ETHAMBUTOL to prophylacticallytreat or prevent an opportunistic Mycobacterium avium complex infection.In another specific embodiment, Therapeutics of the invention are usedin any combination with RIFABUTIN, CLARITHROMYCIN, and/or AZITHROMYCINto prophylactically treat or prevent an opportunistic Mycobacteriumtuberculosis infection. In another specific embodiment, Therapeutics ofthe invention are used in any combination with GANCICLOVIR, FOSCARNET,and/or CIDOFOVIR to prophylactically treat or prevent an opportunisticcytomegalovirus infection. In another specific embodiment, Therapeuticsof the invention are used in any combination with FLUCONAZOLE,ITRACONAZOLE, and/or KETOCONAZOLE to prophylactically treat or preventan opportunistic fungal infection. In another specific embodiment,Therapeutics of the invention are used in any combination with ACYCLOVIRand/or FAMCICOLVIR to prophylactically treat or prevent an opportunisticherpes simplex virus type I and/or type II infection. In anotherspecific embodiment, Therapeutics of the invention are used in anycombination with PYRIMETHAMINE and/or LEUCOVORIN to prophylacticallytreat or prevent an opportunistic Toxoplasma -gondii infection. Inanother specific embodiment, Therapeutics of the invention are used inany combination with LEUCOVORIN and/or NEUPOGEN to prophylacticallytreat or prevent an opportunistic bacterial infection.

In a further embodiment, the Therapeutics of the invention areadministered in combination with an antiviral agent. Antiviral agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine.

In a further embodiment, the Therapeutics of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, amoxicillin, beta-lactamases, aminoglycosides,beta-lactam (glycopeptide), beta-lactamases, Clindamycin,chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin,erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins,quinolones, rifampin, streptomycin, sulfonamide, tetracyclines,trimethoprim, trimethoprim-sulfamthoxazole, and vancomycin.

Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the Therapeutics of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells.

In specific embodiments, Therapeutics of the invention are administeredin combination with immunosuppressants. Immunosuppressants preparationsthat may be administered with the Therapeutics of the invention include,but are not limited to, ORTHOCLONE (OKT3), SANDIMMUNE/NEORAL/SANGDYA(cyclosporin), PROGRAF (tacrolimus), CELLCEPT (mycophenolate),Azathioprine, glucorticosteroids, and RAPAMUNE (sirolimus). In aspecific embodiment, immunosuppressants may be used to prevent rejectionof organ or bone marrow transplantation.

In an additional embodiment, Therapeutics of the invention areadministered alone or in combination with one or more intravenous immuneglobulin preparations. Intravenous immune globulin preparations that maybe administered with the Therapeutics of the invention include, but notlimited to, GAMMAR, IVEEGAM, SANDOGLOBULIN, GAMMAGARD S/D, and GAMIMUNE.In a specific embodiment, Therapeutics of the invention are administeredin combination with intravenous immune globulin preparations intransplantation therapy (e.g., bone marrow transplant).

In an additional embodiment, the Therapeutics of the invention areadministered alone or in combination with an anti-inflammatory agent.Anti-inflammatory agents that may be administered with the Therapeuticsof the invention include, but are not limited to, glucocorticoids andthe nonsteroidal anti-inflammatories, aminoarylcarboxylic acidderivatives, arylacetic acid derivatives, arylbutyric acid derivatives,arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,pyrazolones, salicylic acid derivatives, thiazinecarboxamides,e-acetamidocaproic acid, S-adenosylmethionine, 3-amino4-hydroxybutyricacid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide,ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein,oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, andtenidap.

In another embodiment, compositions of the invention are administered incombination with a chemotherapeutic agent. Chemotherapeutic agents thatmay be administered with the Therapeutics of the invention include, butare not limited to, antibiotic derivatives (e.g., doxorubicin,bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g.,tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate,floxuridine, interferon alpha-2b, glutamic acid, plicamycin,mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine,BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide,estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

In a specific embodiment, Therapeutics of the invention are administeredin combination with CHOP (cyclophosphamide, doxorubicin, vincristine,and prednisone) or any combination of the components of CHOP. In anotherembodiment, Therapeutics of the invention are administered incombination with Rituximab. In a further embodiment, Therapeutics of theinvention are administered with Rituxmab and CHOP, or Rituxmab and anycombination of the components of CHOP.

In an additional embodiment, the Therapeutics of the invention areadministered in combination with cytokines. Cytokines that may beadministered with the Therapeutics of the invention include, but are notlimited to, IL2, IL3, IL4, IL5, IL6, IL7, EL10, IL12, IL13, IL15,anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment,Therapeutics of the invention may be administered with any interleukin,including, but not limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4,IL-S, IL-6, IL-7, L-8, IL-9 IL-10, IL-11, IL-12, IL-13, IL,14, IL-15,IL-16, IL-17, IL-18, IL-19, IL-20, and IL-21.

In an additional embodiment, the Therapeutics of the invention areadministered in combination with angiogenic proteins. Angiogenicproteins that may be administered with the Therapeutics of the inventioninclude, but are not limited to, Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-682110;Platelet Derived Growth Factor-B (P)DGF-B), as disclosed in EuropeanPatent Number EP-2823 17; Placental Growth Factor (PlGF), as disclosedin International Publication Number WO 92/06194; Placental GrowthFactor-2 (PlGF-2), as disclosed in Hauser et al., Gorwth Factors,4:259-268 (1993); Vascular Endothelial Growth Factor (VEGF), asdisclosed in International Publication Number WO 90/13649; VascularEndothelial Growth Factor-A (VEGF-A), as disclosed in European PatentNumber EP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), asdisclosed in International Publication Number WO 96/39515; VascularEndothelial Growth Factor B (VEGF-3); Vascular Endothelial Growth FactorB-1 86 (VEGF-B186), as disclosed in International Publication Number WO96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/02543; Vascular EndothelialGrowth Factor-D (VEGF-D), as disclosed in International PublicationNumber WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E),as disclosed in German Patent Number DE19639601. The above mentionedreferences are incorporated herein by reference herein.

In an additional embodiment, the Therapeutics of the invention areadministered in combination with hematopoietic growth factors.Hematopoietic growth factors that may be administered with theTherapeutics of the invention include, but are not limited to, LEUKINE(SARGRAMOSTIM) and NEUPOGEN (FILGRASTIM).

In an additional embodiment, the Therapeutics of the invention areadministered in combination with Fibroblast Growth Factors. FibroblastGrowth Factors that may be administered with the Therapeutics of theinvention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF4,FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13,FGF-14, and FGF-15.

In a specific embodiment, formulations of the present invention mayfurther comprise antagonists of P-glycoprotein (also referred to as themultiresistance protein, or PGP), including antagonists of its encodingpolynucleotides (e.g., antisense oligonucleotides, ribozymes,zinc-finger proteins, etc.). P-glycoprotein is well known for decreasingthe efficacy of various drug administrations due to its ability toexport intracellular levels of absorbed drug to the cell exterior. Whilethis activity has been particularly pronounced in cancer cells inresponse to the administration of chemotherapy regimens, a variety ofother cell types and the administration of other drug classes have beennoted (e.g., T-cells and anti-HIV drugs). In fact, certain mutations inthe PGP gene significantly reduces PGP function, making it less able toforce drugs out of cells. People who have two versions of the mutatedgene—one inherited from each parent—have more than four times less PGPthan those with two normal versions of the gene. People may also haveone normal gene and one mutated one. Certain ethnic populations haveincreased incidence of such PGP mutations. Among individuals from Ghana,Kenya, the Sudan, as well as African Americans, frequency of the normalgene ranged from 73% to 84%. In contrast, the frequency was 34% to 59%among British whites, Portuguese, Southwest Asian, Chinese, Filipino andSaudi populations. As a result, certain ethnic populations may requireincreased administration of PGP antagonist in the formulation of thepresent invention to arrive at the an efficacious dose of thetherapeutic (e.g., those from African descent). Conversely, certainethnic populations, particularly those having increased frequency of themutated PGP (e.g., of Caucasian descent, or non-African descent) mayrequire less pharmaceutical compositions in the formulation due to aneffective increase in efficacy of such compositions as a result of theincreased effective absorption (e.g., less PGP activity) of saidcomposition.

Moreover, in another specific embodiment, formulations of the presentinvention may further comprise antagonists of OATP2 (also referred to asthe multiresistance protein, or MRP2), including antagonists of itsencoding polynucleotides (e.g., antisense oligonucleotides, ribozymes,zinc-finger proteins, etc.). The invention also further comprises anyadditional antagonists known to inhibit proteins thought to beattributable to a multidrug resistant phenotype in proliferating cells.

Preferred antagonists that formulations of the present may compriseinclude the potent P-glycoprotein inhibitor elacridar, and/or LY-335979.Other P-glycoprotein known in the art are also encompassed by thepresent invention.

In additional embodiments, the Therapeutics of the invention areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

Example 27 Method of Treating Decreased Levels of the Polypeptide

The present invention relates to a method for treating an individual inneed of an increased level of a polypeptide of the invention in the bodycomprising administering to such an individual a composition comprisinga therapeutically effective amount of an agonist of the invention(including polypeptides of the invention). Moreover, it will beappreciated that conditions caused by a decrease in the standard ornormal expression level of a secreted protein in an individual can betreated by administering the polypeptide of the present invention,preferably in the secreted form. Thus, the invention also provides amethod of treatment of an individual in need of an increased level ofthe polypeptide comprising administering to such an individual aTherapeutic comprising an amount of the polypeptide to increase theactivity level of the polypeptide in such an individual.

For example, a patient with decreased levels of a polypeptide receives adaily dose 0.1-100 ug/kg of the polypeptide for six consecutive days.Preferably, the polypeptide is in the secreted form. The exact detailsof the dosing scheme, based on administration and formulation, areprovided herein.

Example 28 Method of Treating Increased Levels of the Polypeptide

The present invention also relates to a method of treating an individualin need of a decreased level of a polypeptide of the invention in thebody comprising administering to such an individual a compositioncomprising a therapeutically effective amount of an antagonist of theinvention (including polypeptides and antibodies of the invention).

In one example, antisense technology is used to inhibit production of apolypeptide of the present invention. This technology is one example ofa method of decreasing levels of a polypeptide, preferably a secretedform, due to a variety of etiologies, such as cancer. For example, apatient diagnosed with abnormally increased levels of a polypeptide isadministered intravenously antisense polynucleotides at 0.5, 1.0, 1.5,2.0 and 3.0 mg/kg day for 21 days. This treatment is repeated after a7-day rest period if the treatment was well tolerated. The formulationof the antisense polynucleotide is provided herein.

Example 29 Method of Treatment Using Gene Therapy-Ex Vivo

One method of gene therapy transplants fibroblasts, which are capable ofexpressing a polypeptide, onto a patient. Generally, fibroblasts areobtained from a subject by skin biopsy. The resulting tissue is placedin tissue-culture medium and separated into small pieces. Small chunksof the tissue are placed on a wet surface of a tissue culture flask,approximately ten pieces are placed in each flask. The flask is turnedupside down, closed tight and left at room temperature over night. After24 hours at room temperature, the flask is inverted and the chunks oftissue remain fixed to the bottom of the flask and fresh media (e.g.,Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added.The flasks are then incubated at 37 degree C. for approximately oneweek.

At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention can beamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively as set forth in Example 13 using primers andhaving appropriate restriction sites and initiation/stop codons, ifnecessary. Preferably, the 5′ primer contains an EcoRI site and the 3′primer includes a HindHI site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is then used totransform bacteria HB101, which are then plated onto agar containingkanamycin for the purpose of confirming that the vector has the gene ofinterest properly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellstransduced with the vector. The packaging cells now produce infectiousviral particles containing the gene (the packaging cells are nowreferred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his. Once the fibroblasts have been efficientlyinfected, the fibroblasts are analyzed to determine whether protein isproduced.

The engineered fibroblasts are then transplanted onto the host, eitheralone or after having been-grown to confluence on cytodex 3microcarrier-beads.

Example 30 Gene Therapy using Endogenous Genes Corresponding toPolynucleotides of the Invention

Another method of gene therapy according to the present inventioninvolves operably associating the endogenous polynucleotide sequence ofthe invention with a promoter via homologous recombination as described,for example, in U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;International Publication NO: WO 96/29411, published Sep. 26, 1996;International Publication NO: WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); andZijlstra et al., Nature, 342:435-438 (1989). This method involves theactivation of a gene which is present in the target cells, but which isnot expressed in the cells, or is expressed at a lower level thandesired.

Polynucleotide constructs are made which contain a promoter andtargeting sequences, which are homologous to the 5′ non-coding sequenceof endogenous polynucleotide sequence, flanking the promoter. Thetargeting sequence will be sufficiently near the 5′ end of thepolynucleotide sequence so the promoter will be operably linked to theendogenous sequence upon homologous recombination. The promoter and thetargeting sequences can be amplified using PCR. Preferably, theamplified promoter contains distinct restriction enzyme sites on the 5′and 3′ ends. Preferably, the 3′ end of the first targeting sequencecontains the same restriction enzyme site as the 5′ end of the amplifiedpromoter and the 5′ end of the second targeting sequence contains thesame restriction site as the 3′ end of the amplified promoter.

The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel then purified by phenol extraction andethanol precipitation.

In this Example, the polynucleotide constructs are administered as nakedpolynucleotides via electroporation. However, the polynucleotideconstructs may also be administered with transfection-facilitatingagents, such as liposomes, viral sequences, viral particles,precipitating agents, etc. Such methods of delivery are known in theart.

Once the cells are transfected, homologous recombination will take placewhich results in the promoter being operably linked to the endogenouspolynucleotide sequence. This results in the expression ofpolynucleotide corresponding to the polynucleotide in the cell.Expression may be detected by immunological staining, or any othermethod known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in DMEM +10% fetal calf serum. Exponentially growing orearly stationary phase fibroblasts are trypsinized and rinsed from theplastic surface with nutrient medium. An aliquot of the cell suspensionis removed for counting, and the remaining cells are subjected tocentrifugation. The supernatant is aspirated and the pellet isresuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137mM NaCl, 5 mM KCl, 0.7 mM Na2 HPO4, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalburnin. The final cell suspension contains approximately 3×106cells/ml. Electroporation should be performed immediately followingresuspension.

Plasmid DNA is prepared according to standard techniques. For example,to construct a plasmid for targeting to the locus corresponding to thepolynucleotide of the invention, plasmid pUC18 (MBI Fermentas, Amherst,N.Y.) is digested with HindIII. The CMV promoter is amplified by PCRwith an XbaI site on the 5′ end and a BamHI site on the 3′end. Twonon-coding sequences are amplified via PCR: one non-coding sequence(fragment 1) is amplified with a HindHI site at the 5′ end and an Xbasite at the 3′end; the other non-coding sequence (fragment 2) isamplified with a BamHI site at the 5′end and a HindIII site at the3′end. The CMV promoter and the fragments (1 and 2) are digested withthe appropriate enzymes (CMV promoter—XbaI and BamHI; fragment 1—XbaI;fragment 2-BamHI) and ligated together. The resulting ligation productis digested with HindIII, and ligated with the HindIII-digested pUC18plasmid.

Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap(Bio-Rad). The final DNA concentration is generally at least 120 μg/ml.0.5 ml of the cell suspension (containing approximately 1.5.×106 cells)is then added to the cuvette, and the cell suspension and DNA solutionsare gently mixed. Electroporation is performed with a Gene-Pulserapparatus (Bio-Rad). Capacitance and voltage are set at 960 PF and250-300 V, respectively. As voltage increases, cell survival decreases,but the percentage of surviving cells that stably incorporate theintroduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 mSec should be observed.

Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37 degree C. The following day, the media isaspirated and replaced with 10 ml of fresh media and incubated for afurther 16-24 hours.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product. The fibroblastscan then be introduced into a patient as described above.

Example 31 Method of Treatment Using Gene Therapy—In Vivo

Another aspect of the present invention is using in vivo gene therapymethods to treat disorders, diseases and conditions. The gene therapymethod relates to the introduction of naked nucleic acid (DNA, RNA, andantisense DNA or RNA) sequences into an animal to increase or decreasethe expression of the polypeptide. The polynucleotide of the presentinvention may be operatively linked to a promoter or any other geneticelements necessary for the expression of the polypeptide by the targettissue. Such gene therapy and delivery techniques and methods are knownin the art, see, for example, WO90/11092, WO98/11779; U.S. Pat. No.5,693,622, 5,705,151, 5,580,859; Tabata et al., Cardiovasc. Res.35(3):470-479 (1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997);Wolff, Neuromuscul. Disord. 7(5):314-318 (1997); Schwartz et al., GeneTher. 3(5):405-411 (1996); Tsurumi et al., Circulation 94(12):3281-3290(1996) (incorporated herein by reference).

The polynucleotide constructs may be delivered by any method thatdelivers injectable materials to the cells of an animal, such as,injection into the interstitial space of tissues (heart, muscle, skin,lung, liver, intestine and the like). The polynucleotide constructs canbe delivered in a pharmaceutically acceptable liquid or aqueous carrier.

The term “naked” polynucleotide, DNA or RNA, refers to sequences thatare free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the polynucleotides of the present invention may alsobe delivered in liposome formulations (such as those taught in FelgnerP. L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. etal. (1995) Biol. Cell 85(1):1-7) which can be prepared by methods wellknown to those skilled in the art.

The polynucleotide vector constructs used in the gene therapy method arepreferably constructs that will not integrate into the host genome norwill they contain sequences that allow for replication. Any strongpromoter known to those skilled in the art can be used for driving theexpression of DNA. Unlike other gene therapies techniques, one majoradvantage of introducing naked nucleic acid sequences into target cellsis the transitory nature of the polynucleotide synthesis in the cells.Studies have shown that non-replicating DNA sequences can be introducedinto cells to provide production of the desired polypeptide for periodsof up to six months.

The polynucleotide construct can be delivered to the interstitial spaceof tissues within the an animal, including of muscle, skin, brain, lung,liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellularfluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

For the naked polynucleotide injection, an effective dosage amount ofDNA or RNA will be in the range of from about 0.05 g/kg body weight toabout 50 mg/kg body weight. Preferably the dosage will be from about0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kgto about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, nakedpolynucleotide constructs can be delivered to arteries duringangioplasty by the catheter used in the procedure.

The dose response effects of injected polynucleotide in muscle in vivois determined as follows. Suitable template DNA for production of mRNAcoding for polypeptide of the present invention is prepared inaccordance with a standard recombinant DNA methodology. The templateDNA, which may be either circular or linear, is either used as naked DNAor complexed with liposomes. The quadriceps muscles of mice are theninjected with various amounts of the template DNA.

Five to six week old female and male Balb/C mice are anesthetized byintraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incisionis made on the anterior thigh, and the quadriceps muscle is directlyvisualized. The template DNA is injected in 0.1 ml of carrier in a I ccsyringe through a 27 gauge needle over one minute, approximately 0.5 cmfrom the distal insertion site of the muscle into the knee and about 0.2cm deep. A suture is placed over the injection site for futurelocalization, and the skin is closed with stainless steel clips.

After an appropriate incubation time (e.g., 7 days) muscle extracts areprepared by excising the entire quadriceps. Every fifth 15 urncross-section of the individual quadriceps muscles is histochemicallystained for protein expression. A time course for protein expression maybe done in a similar fashion except that quadriceps from different miceare harvested at different times. Persistence of DNA in muscle followinginjection may be determined by Southern blot analysis after preparingtotal cellular DNA and HIRT supernatants from injected and control mice.The results of the above experimentation in mice can be use toextrapolate proper dosages and other treatment parameters in humans andother animals using naked DNA.

Example 32 Transgenic Animals

The polypeptides of the invention can also be expressed in transgenicanimals. Animals of any species, including, but not limited to, mice,rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep,cows and non-human primates, e.g., baboons, monkeys, and chimpanzees maybe used to generate transgenic animals. In a specific embodiment,techniques described herein or otherwise known in the art, are used toexpress polypeptides of the invention in humans, as part of a genetherapy protocol.

Any technique known in the art may be used to introduce the transgene(i.e., polynucleotides of the invention) into animals to produce thefounder lines of transgenic animals. Such techniques include, but arenot limited to, pronuclear microinjection (Paterson et al., Appl.Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology(NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834(1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirusmediated gene transfer into germ lines (Van der Putten et al., Proc.Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; genetargeting in embryonic stem cells (Thompson et al., Cell 56:313-321(1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814 (1983)); introduction of the polynucleotides of theinvention using a gene gun (see, e.g., Ulmer et al., Science 259:1745(1993); introducing nucleic acid constructs into embryonic pleuripotentstem cells and transferring the stem cells back into the blastocyst; andsperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989);etc. For a review of such techniques, see Gordon, “Transgenic Animals,”Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by referenceherein in its entirety.

Any technique known in the art may be used to produce transgenic clonescontaining polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric. The transgene may be integrated as a single transgene or asmultiple copies such as in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA89:6232-6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide transgene be integrated into thechromosomal site of the endogenous gene, gene targeting is preferred.Briefly, when such a technique is to be utilized, vectors containingsome nucleotide sequences homologous to the endogenous gene are designedfor the purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of thenucleotide sequence of the endogenous gene. The transgene may also beselectively introduced into a particular cell type, thus inactivatingthe endogenous gene in only that cell type, by following, for example,the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). Theregulatory sequences required for such a cell-type specific inactivationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art.

Once transgenic animals have been generated, the expression of therecombinant gene may be assayed utilizing standard techniques. Initialscreening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR(RT-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

Transgenic animals of the invention have uses which include, but are notlimited to, animal model systems useful in elaborating the biologicalfunction of polypeptides of the present invention, studying diseases,disorders, and/or conditions associated with aberrant expression, and inscreening for compounds effective in ameliorating such diseases,disorders, and/or conditions.

Example 33 Knock-Out Animals

Endogenous gene expression can also be reduced by inactivating or“knocking out” the gene and/or its promoter using targeted homologousrecombination. (E.g., see Smithies et al., Nature 317:230-234 (1985);Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell5:313-321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene (e.g., see Thomas & Capecchi1987 and Thompson 1989, supra). However this approach can be routinelyadapted for use in humans provided the recombinant DNA constructs aredirectly administered or targeted to the required site in vivo usingappropriate viral vectors that will be apparent to those of skill in theart.

In further embodiments of the invention, cells that are geneticallyengineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.,lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cellsare genetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe polypeptides of the invention. The engineered cells which expressand preferably secrete the polypeptides of the invention can beintroduced into the patient systemically, e.g., in the circulation, orintraperitoneally.

Alternatively, the cells can be incorporated into a matrix and implantedin the body, e.g., genetically engineered fibroblasts can be implantedas part of a skin graft; genetically engineered endothelial cells can beimplanted as part of a lymphatic or vascular graft. (See, for example,Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S.Pat. No. 5,460,959 each of which is incorporated by reference herein inits entirety).

When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

Transgenic and “knock-out” animals of the invention have uses whichinclude, but are not limited to, animal model systems useful inelaborating the biological function of polypeptides of the presentinvention, studying diseases, disorders, and/or conditions associatedwith aberrant expression, and in screening for compounds effective inameliorating such diseases, disorders, and/or conditions.

Example 34 Method of Isolating Antibody Fragments Directed AgainstCAN-12 From a Library of scFvs

Naturally occurring V-genes isolated from human PBLs are constructedinto a library of antibody fragments which contain reactivities againstCAN-12 to which the donor may or may not have been exposed (see e.g.,U.S. Pat. No. 5,885,793 incorporated herein by reference in itsentirety).

Rescue of the Library. A library of scFvs is constructed from the RNA ofhuman PBLs as described in PCT publication WO 92/01047. To rescue phagedisplaying antibody fragments, approximately 109 E. coli harboring thephagemid are used to inoculate 50 ml of 2×TY containing 1% glucose and100 μg/ml of ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8 withshaking. Five ml of this culture is used to inoculate 50 ml of2×TY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene III, seePCT publication WO 92/01047) are added and the culture incubated at 37°C. for 45 minutes without shaking and then at 37° C. for 45 minutes withshaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and thepellet resuspended in 2 liters of 2×TY containing 100 μg/ml ampicillinand 50 ug/ml kanamycin and grown overnight. Phage are prepared asdescribed in PCT publication WO 92/01047.

M13 delta gene III is prepared as follows: M13 delta gene El[ helperphage does not encode gene Ell protein, hence the phage(mid) displayingantibody fragments have a greater avidity of binding to antigen.Infectious M13 delta gene III particles are made by growing the helperphage in cells harboring a pUC19 derivative supplying the wild type geneIII protein during phage morphogenesis. The culture is incubated for 1hour at 37° C. without shaking and then for a further hour at 37° C.with shaking. Cells are spun down (EEC-Centra 8,400 r.p.m. for 10 min),resuspended in 300 ml 2×TY broth containing 100 μg ampicillin/ml and 25μg kanamycin/ml (2×TY-AMP-KAN) and grown overnight, shaking at 37° C.Phage particles are purified and concentrated from the culture medium bytwo PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBSand passed through a 0.45 μm filter (Minisart NML; Sartorius) to give afinal concentration of approximately 1013 transducing units/ml(ampicillin-resistant clones).

Panning of the Library. Immunotubes (Nunc) are coated overnight in PBSwith 4 ml of either 100 μg/ml or 10 μg/ml of a polypeptide of thepresent invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at37° C. and then washed 3 times in PBS. Approximately 1013 TU of phage isapplied to the tube and incubated for 30 minutes at room temperaturetumbling on an over and under turntable and then left to stand foranother 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and10 times with PBS. Phage are eluted by adding 1 ml of 100 mMtriethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 ml of 1.0MTris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coliTG1 by incubating eluted phage with bacteria for 30 minutes at 37° C.The E. coli are then plated on TYE plates containing 1% glucose and 100μg/ml ampicillin. The resulting bacterial library is then rescued withdelta gene 3 helper phage as described above to prepare phage for asubsequent round of selection. This process is then repeated for a totalof 4 rounds of affinity purification with tube-washing increased to 20times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders. Eluted phage from the 3rd and 4th rounds ofselection are used to infect E. coli HB 2151 and soluble scFv isproduced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 pg/ml of thepolypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clonespositive in ELISA are further characterized by PCR fingerprinting (see,e.g., PCT publication WO 92/01047) and then by sequencing. These ELISApositive clones may also be further characterized by techniques known inthe art, such as, for example, epitope mapping, binding affinity,receptor signal transduction, ability to block or competitively inhibitantibody/antigen binding, and competitive agonistic or antagonisticactivity.

Moreover, in another preferred method, the antibodies directed againstthe polypeptides of the present invention may be produced in plants.Specific methods are disclosed in U.S. Pat. Nos. 5,959,177, and6,080,560, which are hereby incorporated in their entirety herein. Themethods not only describe methods of expressing antibodies, but also themeans of assembling foreign multimeric proteins in plants (i.e.,antibodies, etc,), and the subsequent secretion of such antibodies fromthe plant.

Example 35 Identification and Cloning of VH and VL domains of AntibodiesDirected Against the CAN-12 Polypeptide

VH and VL domains may be identified and cloned from cell linesexpressing an antibody directed against a CAN-12 epitope by performingPCR with VH and VL specific primers on cDNA made from the antibodyexpressing cell lines. Briefly, RNA is isolated from the cell lines andused as a template for RT-PCR designed to amplify the VH and VL domainsof the antibodies expressed by the EBV cell lines. Cells may be lysedusing the TRIzol reagent (Life Technologies, Rockville, Md.) andextracted with one fifth volume of chloroform. After addition ofchloroform, the solution is allowed to incubate at room temperature for10 minutes, and then centrifuged at 14,000 rpm for 15 minutes at 4 C ina tabletop centrifuge. The supernatant is collected and RNA isprecipitated using an equal volume of isopropanol. Precipitated RNA ispelleted by centrifuging at 14,000 rpm for 15 minutes at 4 C in atabletop centrifuge.

Following centrifugation, the supernatant is discarded and washed with75% ethanol. Following the wash step, the RNA is centrifuged again at800 rpm for 5 minutes at 4 C. The supernatant is discarded and thepellet allowed to air dry. RNA is the dissolved in DEPC water and heatedto 6° C. for 10 minutes. Quantities of RNA can be determined usingoptical density measurements. cDNA may be synthesized, according tomethods well-known in the art and/or described herein, from 1.5-2.5micrograms of RNA using reverse transciptase and random hexamer primers.cDNA is then used as a template for PCR amplification of VH and VLdomains.

Primers used to amplify VH and VL genes are shown below. Typically a PCRreaction makes use of a single 5′primer and a single 3′primer.Sometimes, when the amount of available RNA template is limiting, or forgreater efficiency, groups of 5′ and/or 3′primers may be used. Forexample, sometimes all five VH-5′primers and all JH3′primers are used ina single PCR reaction. The PCR reaction is carried out in a 50microliter volume containing 1× PCR buffer, 2 mM of each DNTP, 0.7 unitsof High Fidelity Taq polymerse, 5′primer mix, 3′primer mix and 7.5microliters of cDNA. The 5′and 3′primer mix of both VH and VL can bemade by pooling together 22 pmole and 28 pmole, respectively, of each ofthe individual primers. PCR conditions are: 96 C for 5 minutes; followedby 25 cycles of 94 C for 1 minute, 5° C. for 1 minute, and 72 C for 1minute; followed by an extension cycle of 72 C for 10 minutes. After thereaction has been completed, sample tubes may be stored at 4 C. PrimerSequences Used to Amplify VH domains. SEQ ID Primer name Primer SequenceNO: Hu VH1 -5′ CAGGTGCAGCTGGTGCAGTCTGG 105 Hu VH2 -5′CAGGTCAACTTAAGGGAGTCTGG 106 Hu VH3 -5′ GAGGTGCAGCTGGTGGAGTCTGG 107 HuVH4 -5′ CAGGTGCAGCTGCAGGAGTCGGG 108 Hu VH5 -5′ GAGGTGCAGCTGTTGCAGTCTGC109 Hu VH6 -5′ CAGGTACAGCTGCAGCAGTCAGG 110 Hu JH1 -5′TGAGGAGACGGTGACCAGGGTGCC 111 Hu JH3 -5′ TGAAGAGACGGTGACCATTGTCCC 112 HuJH4 -5′ TGAGGAGACGGTGACCAGGGTTCC 113 Hu JH6 -5′ TGAGGAGACGGTGACCGTGGTCCC114 Hu Vkappa1 -5′ GACATCCAGATGACCCAGTCTCC 115 Hu Vkappa2a -5′GATGTTGTGATGACTCAGTCTCC 116 Hu Vkappa2b -5′ GATATTGTGATGACTCAGTCTCC 117Hu Vkappa3 -5′ GAAATTGTGTTGACGCAGTCTCC 118 Hu Vkappa4 -5′GACATCGTGATGACCCAGTCTCC 119 Hu Vkappa5 -5′ GAAACGACACTCACGCAGTCTCC 120Hu Vkappa6 -5′ GAAATTGTGCTGACTCAGTCTCC 121 Hu Vlambda1 -5′CAGTCTGTGTTGACGCAGCCGCC 122 Hu Vlambda2 -5′ CAGTCTGCCCTGACTCAGCCTGC 123Hu Vlambda3 -5′ TCCTATGTGCTGACTCAGCCACC 124 Hu Vlambda3b -5′TCTTCTGAGCTGACTCAGGACCC 125 Hu Vlambda4 -5′ CACGTTATACTGACTCAACCGCC 126Hu Vlambda5 -5′ CAGGCTGTGCTCACTCAGCCGTC 127 Hu Vlambda6 -5′AATTTTATGCTGACTCAGCCCCA 128 Hu Jkappa1 -3′ ACGTTTGATTTCCACCTTGGTCCC 129Hu Jkappa2 -3′ ACGTTTGATCTCCAGCTTGGTCCC 130 Hu Jkappa3 -3′ACGTTTGATATCCACTTTGGTCCC 131 Hu Jkappa4 -3′ ACGTTTGATCTCCACCTTGGTCCC 132Hu Jkappa5 -3′ ACGTTTAATCTCCAGTCGTGTCCC 133 Hu Vlambda1 -3′CAGTCTGTGTTGACGCAGCCGCC 134 Hu Vlambda2 -3′ CAGTCTGCCCTGACTCAGCCTGC 135Hu Vlambda3 -3′ TCCTATGTGCTGACTCAGCCACC 136 Hu Vlambda3b -3′TCTTCTGAGCTGACTCAGGACCC 137 Hu Vlambda4 -3′ CACGTTATACTGACTCAACCGCC 138Hu Vlambda5 -3′ CAGGCTGTGCTCACTCAGCCGTC 139 Hu Vlambda6 -3′AATTTTATGCTGACTCAGCCCCA 140

PCR samples are then electrophoresed on a 1.3% agarose gel. DNA bands ofthe expected sizes (−506 base pairs for VH domains, and 344 base pairsfor VL domains) can be cut out of the gel and purified using methodswell known in the art and/or described herein.

Purified PCR products can be ligated into a PCR cloning vector (TAvector from Invitrogen Inc., Carlsbad, Calif.). Individual cloned PCRproducts can be isolated after transfection of E. coli and blue/whitecolor selection. Cloned PCR products may then be sequenced using methodscommonly known in the art and/or described herein.

The PCR bands containing the VH domain and the VL domains can also beused to create full-length Ig expression vectors. VH and VL domains canbe cloned into vectors containing the nucleotide sequences of a heavy(e.g., human IgG1 or human IgG4) or light chain (human kappa or humanambda) constant regions such that a complete heavy or light chainmolecule could be expressed from these vectors when transfected into anappropriate host cell. Further, when cloned heavy and light chains areboth expressed in one cell line (from either one or two vectors), theycan assemble into a complete functional antibody molecule that issecreted into the cell culture medium. Methods using polynucleotidesencoding VH and VL antibody domain to generate expression vectors thatencode complete antibody molecules are well known within the art.

Example 36 Assays Detecting Stimulation or Inhibition of B cellProliferation and Differentiation

Generation of functional humoral immune responses requires both solubleand cognate signaling between B-lineage cells and theirmicroenvironment. Signals may impart a positive stimulus that allows aB-lineage cell to continue its programmed development, or a negativestimulus that instructs the cell to arrest its current developmentalpathway. To date, numerous stimulatory and inhibitory signals have beenfound to influence B cell responsiveness including IL-2, IL-4, IL-5,IL-6, IL-7, IL-10, IL-13, IL-14 and IL-15. Interestingly, these signalsare by themselves weak effectors but can, in combination with variousco-stimulatory proteins, induce activation, proliferation,differentiation, homing, tolerance and death among B cell populations.

One of the best studied classes of B-cell co-stimulatory proteins is theTNF-superfamily. Within this family CD40, CD27, and CD30 along withtheir respective higands CD154, CD70, and CD153 have been found toregulate a variety of immune responses. Assays which allow for thedetection and/or observation of the proliferation and differentiation ofthese B-cell populations and their precursors are valuable tools indetermining the effects various proteins may have on these B-cellpopulations in terms of proliferation and differentiation. Listed beloware two assays designed to allow for the detection of thedifferentiation, proliferation, or inhibition of B-cell populations andtheir precursors.

In Vitro Assay—Purified polypeptides of the invention, or truncatedforms thereof, is assessed for its ability to induce activation,proliferation, differentiation or inhibition and/or death in B-cellpopulations and their precursors. The activity of the polypeptides ofthe invention on purified human tonsillar B cells, measuredqualitatively over the dose range from 0.1 to 10,000 ng/mL, is assessedin a standard B-lymphocyte co-stimulation assay in which purifiedtonsillar B cells are cultured in the presence of either formalin-fixedStaphylococcus aureus Cowan I (SAC) or immobilized anti-human IgMantibody as the priming agent. Second signals such as IL-2 and IL-15synergize with SAC and IgM crosslinking to elicit B cell proliferationas measured by tritiated-thymidine incorporation. Novel synergizingagents can be readily identified using this assay. The assay involvesisolating human tonsillar B cells by magnetic bead (MACS) depletion ofCD3-positive cells. The resulting cell population is greater than 95% Bcells as assessed by expression of CD45R(B220).

Various dilutions of each sample are placed into individual wells of a96-well plate to which are added 105 B-cells suspended in culture medium(RPMI 1640 containing 10% FBS, 5×10-5M 2ME, 100 U/ml penicillin, 10ug/ml streptomycin, and 10-5 dilution of SAC) in a total volume of 150ul. Proliferation or inhibition is quantitated by a 20 h pulse (1uCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72 h post factoraddition. The positive and negative controls are IL2 and mediumrespectively.

In Vivo Assay—BALB/c mice are injected (i.p.) twice per day with bufferonly, or 2 mg/Kg of a polypeptide of the invention, or truncated formsthereof. Mice receive this treatment for 4 consecutive days, at whichtime they are sacrificed and various tissues and serum collected foranalyses. Comparison of H&E sections from normal spleens and spleenstreated with polypeptides of the invention identify the results of theactivity of the polypeptides on spleen cells, such as the diffusion ofperi-arterial lymphatic sheaths, and/or significant increases in thenucleated cellularity of the red pulp regions, which may indicate theactivation of the differentiation and proliferation of B-cellpopulations. Immunohistochemical studies using a B cell marker,anti-CD45R(B220), are used to determine whether any physiologicalchanges to splenic cells, such as splenic disorganization, are due toincreased B-cell representation within loosely defined B-cell zones thatinfiltrate established T-cell regions.

Flow cytometric analyses of the spleens from mice treated withpolypeptide is used to indicate whether the polypeptide specificallyincreases the proportion of ThB+, CD45R(B220)dull B cells over thatwhich is observed in control mice.

Likewise, a predicted consequence of increased mature B-cellrepresentation in vivo is a relative increase in serum Ig titers.Accordingly, serum IgM and IgA levels are compared between buffer andpolypeptide-treated mice.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 37 T Cell Proliferation Assay

A CD3-induced proliferation assay is performed on PBMCs and is measuredby the uptake of 3H-thymidine. The assay is performed as follows.Ninety-six well plates are coated with 100 (l/well of mAb to CD3 (HIT3a,Pharmingen) or isotype-matched control mAb (B33.1) overnight at 4degrees C. (1 (g/ml in 0.05M bicarbonate buffer, pH 9.5), then washedthree times with PBS. PBMC are isolated by F/H gradient centrifugationfrom human peripheral blood and added to quadruplicate wells(5×104/well) of mAb coated plates in RPMI containing 10% FCS and P/S inthe presence of varying concentrations of polypeptides of the invention(total volume 200 ul). Relevant protein buffer and medium alone arecontrols. After 48 hr. culture at 37 degrees C., plates are spun for 2min. at 1000 rpm and 100 (1 of supernatant is removed and stored −20degrees C. for measurement of IL-2 (or other cytokines) if effect onproliferation is observed. Wells are supplemented with 100 ul of mediumcontaining 0.5 uCi of 3H-thymidine and cultured at 37 degrees C. for18-24 hr. Wells are harvested and incorporation of 3H-thymidine used asa measure of proliferation. Anti-CD3 alone is the positive control forproliferation. IL-2 (100 U/ml) is also used as a control which enhancesproliferation. Control antibody which does not induce proliferation of Tcells is used as the negative controls for the effects of polypeptidesof the invention.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 38 Effect of Polypeptides of the Invention on the Expression ofMHC Class II, Costimulatory and Adhesion Molecules and CellDifferentiation of Monocytes and Monocyte-Derived Human Dendritic Cells

Dendritic cells are generated by the expansion of proliferatingprecursors found in the peripheral blood: adherent PBMC or elutriatedmonocytic fractions are cultured for 7-10 days with GM-CSF (50 ng/ml)and IL4 (20 ng/ml). These dendritic cells have the characteristicphenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHCclass II antigens). Treatment with activating factors, such as TNF-,causes a rapid change in surface phenotype (increased expression of MHCclass I and II, costimulatory and adhesion molecules, downregulation ofFC(RII, upregulation of CD83). These changes correlate with increasedantigen-presenting capacity and with functional maturation of thedendritic cells.

FACS analysis of surface antigens is performed as follows. Cells aretreated 1-3 days with increasing concentrations of polypeptides of theinvention or LPS (positive control), washed with PBS containing 1% BSAand 0.02 mM sodium azide, and then incubated with 1:20 dilution ofappropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at4 degrees C. After an additional wash, the labeled cells are analyzed byflow cytometry on a FACScan (Becton Dickinson).

Effect on the production of cytokines. Cytokines generated by dendriticcells, in particular IL-12, are important in the initiation of T-celldependent immune responses. IL-12 strongly influences the development ofTh1 helper T-cell immune response, and induces cytotoxic T and NK cellfunction. An ELISA is used to measure the IL-12 release as follows.Dendritic cells (106/ml) are treated with increasing concentrations ofpolypeptides of the invention for 24 hours. LPS (100 ng/ml) is added tothe cell culture as positive control. Supernatants from the cellcultures are then collected and analyzed for IL-12 content usingcommercial ELISA kit(e.g., R & D Systems (Minneapolis, Minn.)). Thestandard protocols provided with the kits are used.

Effect on the expression of MHC Class II, costimulatory and adhesionmolecules. Three major families of cell surface antigens can beidentified on monocytes: adhesion molecules, molecules involved inantigen presentation, and Fc receptor. Modulation of the expression ofMHC class II antigens and other costimulatory molecules, such as B7 andICAM-1, may result in changes in the antigen presenting capacity ofmonocytes and ability to induce T cell activation. Increase expressionof Fc receptors may correlate with improved monocyte cytotoxic activity,cytokine release and phagocytosis.

FACS analysis is used to examine the surface antigens as follows.Monocytes are treated 1-5 days with increasing concentrations ofpolypeptides of the invention or LPS (positive control), washed with PBScontaining 1% BSA and 0.02 mM sodium azide, and then incubated with 1:20dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30minutes at 4 degrees C. After an additional wash, the labeled cells areanalyzed by flow cytometry on a FACScan (Becton Dickinson).

Monocyte activation and/or increased survival. Assays for molecules thatactivate (or alternatively, inactivate) monocytes and/or increasemonocyte survival (or alternatively, decrease monocyte survival) areknown in the art and may routinely be applied to determine whether amolecule of the invention functions as an inhibitor or activator ofmonocytes. Polypeptides, agonists, or antagonists of the invention canbe screened using the three assays described below. For each of theseassays, Peripheral blood mononuclear cells (PBMC) are purified fromsingle donor leukopacks (American Red Cross, Baltimore, Md.) bycentrifugation through a Histopaque gradient (Sigma). Monocytes areisolated from PBMC by counterflow centrifugal elutriation.

Monocyte Survival Assay. Human peripheral blood monocytes progressivelylose viability when cultured in absence of serum or other stimuli. Theirdeath results from internally regulated process (apoptosis). Addition tothe culture of activating factors, such as TNF-alpha dramaticallyimproves cell survival and prevents DNA fragmentation. Propidium iodide(PI) staining is used to measure apoptosis as follows. Monocytes arecultured for 48 hours in polypropylene tubes in serum-free medium(positive control), in the presence of 100 ng/ml TNF-alpha (negativecontrol), and in the presence of varying concentrations of the compoundto be tested. Cells are suspended at a concentration of 2×106/ml in PBScontaining PI at a final concentration of 5 (g/ml, and then incubated atroom temperature for 5 minutes before FACScan analysis. PI uptake hasbeen demonstrated to correlate with DNA fragmentation in thisexperimental paradigm.

Effect on cytokine release. An important function ofmonocytes/macrophages is their regulatory activity on other cellularpopulations of the immune system through the release of cytokines afterstimulation. An ELISA to measure cytokine release is performed asfollows. Human monocytes are incubated at a density of 5×105 cells/mlwith increasing concentrations of the a polypeptide of the invention andunder the same conditions, but in the absence of the polypeptide. ForIL-12 production, the cells are primed overnight with IFN (100 U/ml) inpresence of a polypeptide of the invention. LPS (10 ng/ml) is thenadded. Conditioned media are collected after 24h and kept frozen untiluse. Measurement of TNF-alpha, IL-10, MCP-1 and IL-8 is then performedusing a commercially available ELISA kit(e.g., R & D Systems(Minneapolis, Minn.)) and applying the standard protocols provided withthe kit.

Oxidative burst. Purified monocytes are plated in 96-w plate at 2-1×105cell/well. Increasing concentrations of polypeptides of the inventionare added to the wells in a total volume of 0.2 ml culture medium (RPMI1640+10% FCS, glutamine and antibiotics). After 3 days incubation, theplates are centrifuged and the medium is removed from the wells. To themacrophage monolayers, 0.2 ml per well of phenol red solution (140 mMNaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mMphenol red and 19 U/ml of HRPO) is added, together with the stimulant(200 nM PMA). The plates are incubated at 37(C for 2 hours and thereaction is stopped by adding 20 μl 1N NaOH per well. The absorbance isread at 610 nm. To calculate the amount of H2O2 produced by themacrophages, a standard curve of a H2O2 solution of known molarity isperformed for each experiment.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 39 Biological Effects of Polypeptides of the Invention

Astrocyte and Neuronal Assays.

Recombinant polypeptides of the invention, expressed in Escherichia coliand purified as described above, can be tested for activity in promotingthe survival, neurite outgrowth, or phenotypic differentiation ofcortical neuronal cells and for inducing the proliferation of glialfibrillary acidic protein immunopositive cells, astrocytes. Theselection of cortical cells for the bioassay is based on the prevalentexpression of FGF-1 and FGF-2 in cortical structures and on thepreviously reported enhancement of cortical neuronal survival resultingfrom FGF-2 treatment. A thymidine incorporation assay, for example, canbe used to elucidate a polypeptide of the invention's activity on thesecells.

Moreover, previous reports describing the biological effects of FGF-2(basic FGF) on cortical or hippocampal neurons in vitro havedemonstrated increases in both neuron survival and neurite outgrowth(Walicke et al., “Fibroblast growth factor promotes survival ofdissociated hippocampal neurons and enhances neurite extension.” Proc.Natl. Acad. Sci. USA 83:3012-3016. (1986), assay herein incorporated byreference in its entirety). However, reports from experiments done onPC-12 cells suggest that these two responses are not necessarilysynonymous and may depend on not only which FGF is being tested but alsoon which receptor(s) are expressed on the target cells. Using theprimary cortical neuronal culture paradigm, the ability of a polypeptideof the invention to induce neurite outgrowth can be compared to theresponse achieved with FGF-2 using, for example, a thymidineincorporation assay.

Fibroblast and Endothelial Cell Assays.

Human lung fibroblasts are obtained from Clonetics (San Diego, Calif.)and maintained in growth media from Clonetics. Dermal microvascularendothelial cells are obtained from Cell Applications (San Diego,Calif.). For proliferation assays, the human lung fibroblasts and dermalmicrovascular endothelial cells can be cultured at 5,000 cells/well in a96-well plate for one day in growth medium. The cells are then incubatedfor one day in 0.1% BSA basal medium. After replacing the medium withfresh 0.1% BSA medium, the cells are incubated with the test proteinsfor 3 days. Alamar Blue (Alamar Biosciences, Sacramento, Calif.) isadded to each well to a final concentration of 10%. The cells areincubated for 4 hr. Cell viability is measured by reading in a CytoFluorfluorescence reader. For the PGE2 assays, the human lung fibroblasts arecultured at 5,000 cells/well in a 96-well plate for one day. After amedium change to 0.1% BSA basal medium, the cells are incubated withFGF-2 or polypeptides of the invention with or without IL-1( for 24hours. The supernatants are collected and assayed for PGE2 by EIA kit(Cayman, Ann Arbor, Mich.). For the IL-6 assays, the human lungfibroblasts are cultured at 5,000 cells/well in a 96-well plate for oneday. After a medium change to 0.1% BSA basal medium, the cells areincubated with FGF-2 or with or without polypeptides of the inventionIL-1 (for 24 hours. The supernatants are collected and assayed for IL-6by ELISA kit (Endogen, Cambridge, Mass.).

Human lung fibroblasts are cultured with FGF-2 or polypeptides of theinvention for 3 days in basal medium before the addition of Alamar Blueto assess effects on growth of the fibroblasts. FGF-2 should show astimulation at 10 -2500 ng/ml which can be used to compare stimulationwith polypeptides of the invention.

Parkinson Models.

The loss of motor function in Parkinson's disease is attributed to adeficiency of striatal dopamine resulting from the degeneration of thenigrostriatal dopaminergic projection neurons. An animal model forParkinson's that has been extensively characterized involves thesystemic administration of 1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine(MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized bymonoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP+) and released.Subsequently, MPP+ is actively accumulated in dopaminergic neurons bythe high-affinity reuptake transporter for dopamine. MPP+ is thenconcentrated in mitochondria by the electrochemical gradient andselectively inhibits nicotidamide adenine disphosphate: ubiquinoneoxidoreductionase (complex I), thereby interfering with electrontransport and eventually generating oxygen radicals.

It has been demonstrated in tissue culture paradigms that FGF-2 (basicFGF) has trophic activity towards nigral dopaminergic neurons (Ferrariet al., Dev. Biol. 1989). Recently, Dr. Unsicker's group hasdemonstrated that administering FGF-2 in gel foam implants in thestriatum results in the near complete protection of nigral dopaminergicneurons from the toxicity associated with MPTP exposure (Otto andUnsicker, J. Neuroscience, 1990).

Based on the data with FGF-2, polypeptides of the invention can beevaluated to determine whether it has an action similar to that of FGF-2in enhancing dopaminergic neuronal survival in vitro and it can also betested in vivo for protection of dopaminergic neurons in the striatumfrom the damage associated with MPTP treatment. The potential effect ofa polypeptide of the invention is first examined in vitro in adopaminergic neuronal cell culture paradigm. The cultures are preparedby dissecting the midbrain floor plate from gestation day 14 Wistar ratembryos. The tissue is dissociated with trypsin and seeded at a densityof 200,000 cells/ctn2 on polyorthinine-laminin coated glass coverslips.The cells are maintained in Dulbecco's Modified Eagle's medium and F12medium containing hormonal supplements (N1). The cultures are fixed withparaformaldehyde after 8 days in vitro and are processed for tyrosinehydroxylase, a specific marker for dopaminergic neurons,immunohistochemical staining. Dissociated cell cultures are preparedfrom embryonic rats. The culture medium is changed every third day andthe factors are also added at that time.

Since the dopaminergic neurons are isolated from animals at gestationday 14, a developmental time which is past the stage when thedopaminergic precursor cells are proliferating, an increase in thenumber of tyrosine hydroxylase immunopositive neurons would represent anincrease in the number of dopaminergic neurons surviving in vitro.Therefore, if a polypeptide of the invention acts to prolong thesurvival of dopaminergic neurons, it would suggest that the polypeptidemay be involved in Parkinson's Disease.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 40 The Effect of Polypeptides of the Invention on the Growth ofVascular Endothelial Cells

On day 1, human umbilical vein endothelial cells (HUVEC) are seeded at2-5×104 cells/35 mm dish density in M199 medium containing 4% fetalbovine serum (FBS), 16 units/ml heparin, and 50 units/ml endothelialcell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the mediumis replaced with M199 containing 10% FBS, 8 units/ml heparin. Apolypeptide having the amino acid sequence of SEQ ID NO:2, and positivecontrols, such as VEGF and basic FGF (bFGF) are added, at varyingconcentrations. On days 4 and 6, the medium is replaced. On day 8, cellnumber is determined with a Coulter Counter.

An increase in the number of HUVEC cells indicates that the polypeptideof the invention may proliferate vascular endothelial cells.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 41 Stimulatory Effect of Polypeptides of the Invention on theProliferation of Vascular Endothelial Cells

For evaluation of mitogenic activity of growth factors, the colorimetricMTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium)assay with the electron coupling reagent PMS (phenazine methosulfate)was performed (CellTiter 96 AQ, Promega). Cells are seeded in a 96-wellplate (5,000 cells/well) in 0.1 mL serum-supplemented medium and areallowed to attach overnight. After serum-starvation for 12 hours in 0.5%FBS, conditions (bFGF, VEGF165 or a polypeptide of the invention in 0.5%FBS) with or without Heparin (8 U/ml) are added to wells for 48 hours.20 mg of MTS/PMS mixture (1:0.05) are added per well and allowed toincubate for 1 hour at 37° C. before measuring the absorbance at 490 nmin an ELISA plate reader. Background absorbance from control wells (somemedia, no cells) is subtracted, and seven wells are performed inparallel for each condition. See, Leak et al. In Vitro Cell. Dev. Biol.30A:512-518 (1994).

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 42 Inhibition of PDGF-induced Vascular Smooth Muscle CellProliferation Stimulatory Effect

HAoSMC proliferation can be measured, for example, by BrdUrdincorporation. Briefly, subconfluent, quiescent cells grown on the4-chamber slides are transfected with CRP or FITC-labeled AT2-3-LP.Then, the cells are pulsed with 10% calf serum and 6 mg/ml BrdUrd. After24 h, immunocytochemistry is performed by using BrdUrd Staining Kit(Zymed Laboratories). In brief, the cells are incubated with thebiotinylated mouse anti-BrdUrd antibody at 4 degrees C. for 2 h afterbeing exposed to denaturing solution and then incubated with thestreptavidin-peroxidase and diaminobenzidine. After counterstaining withhematoxylin, the cells are mounted for microscopic examination, and theBrdUrd-positive cells are counted. The BrdUrd index is calculated as apercent of the BrdUrd-positive cells to the total cell number. Inaddition, the simultaneous detection of the BrdUrd staining (nucleus)and the FITC uptake (cytoplasm) is performed for individual cells by theconcomitant use of bright field illumination and dark field-UVfluorescent illumination. See, Hayashida et al., J. Biol. Chem.6:271(36):21985-21992 (1996).

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 43 Stimulation of Nitric Oxide Production by Endothelial Cells

Nitric oxide released by the vascular endothelium is believed to be amediator of vascular endothelium relaxation. Thus, activity of apolypeptide of the invention can be assayed by determining nitric oxideproduction by endothelial cells in response to the polypeptide.

Nitric oxide is measured in 96-well plates of confluent microvascularendothelial cells after 24 hours starvation and a subsequent 4 hrexposure to various levels of a positive control (such as VEGF-1) andthe polypeptide of the invention. Nitric oxide in the medium isdetermined by use of the Griess reagent to measure total nitrite afterreduction of nitric oxide-derived nitrate by nitrate reductase. Theeffect of the polypeptide of the invention on nitric oxide release isexamined on HUVEC.

Briefly, NO release from cultured HUVEC monolayer is measured with aNO-specific polarographic electrode connected to a NO meter (Iso-NO,World Precision Instruments Inc.) (1049). Calibration of the NO elementsis performed according to the following equation:2 KNO2+2 KI+2 H2SO4 6 2 NO+I2+2 H2O+2 K2SO4

The standard calibration curve is obtained by adding gradedconcentrations of KNO2 (0, 5, 10, 25, 50, 100, 250, and 500 mmol) intothe calibration solution containing KI and H2S04. The specificity of theIso-NO electrode to NO is previously determined by measurement of NOfrom authentic NO gas (1050). The culture medium is removed and HUVECsare washed twice with Dulbecco's phosphate buffered saline. The cellsare then bathed in 5 ml of filtered Krebs-Henseleit solution in 6-wellplates, and the cell plates are kept on a slide warmer (Lab LineInstruments Inc.) To maintain the temperature at 37° C. The NO sensorprobe is inserted vertically into the wells, keeping the tip of theelectrode 2 mm under the surface of the solution, before addition of thedifferent conditions. S-nitroso acetyl penicillamin (SNAP) is used as apositive control. The amount of released NO is expressed as picomolesper 1×106 endothelial cells. All values reported are means of four tosix measurements in each group (number of cell culture wells). See, Leaket al. Biochem. and Biophys. Res. Comm. 217:96-105 (1995).

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 44 Rescue of Ischemia in Rabbit Lower Limb Model

To study the in vivo effects of polynucleotides and polypeptides of theinvention on ischemia, a rabbit hindlimb ischemia model is created bysurgical removal of one femoral arteries as described previously(Takeshita et al., Am J. Pathol 147:1649-1660 (1995)). The excision ofthe femoral artery results in retrograde propagation of thrombus andocclusion of the external iliac artery. Consequently, blood flow to theischemic limb is dependent upon collateral vessels originating from theinternal iliac artery (Takeshitaet al. Am J. Pathol 147:1649-1660(1995)). An interval of 10 days is allowed for post-operative recoveryof rabbits and development of endogenous collateral vessels. At 10 daypost-operatively (day 0), after performing a baseline angiogram, theinternal iliac artery of the ischemic limb is transfected with 500 mgnaked expression plasmid containing a polynucleotide of the invention byarterial gene transfer technology using a hydrogel-coated ballooncatheter as described (Riessen et al. Hum Gene Ther. 4:749-758 (1993);Leclerc et al. J. Clin. Invest. 90: 936-944 (1992)). When a polypeptideof the invention is used in the treatment, a single bolus of 500 mgpolypeptide of the invention or control is delivered into the internaliliac artery of the ischemic limb over a period of 1 min. through aninfusion catheter. On day 30, various parameters are measured in theserabbits: (a) BP ratio—The blood pressure ratio of systolic pressure ofthe ischemic limb to that of normal limb; (b) Blood Flow and FlowReserve—Resting FL: the blood flow during undilated condition and MaxFL: the blood flow during fully dilated condition (also an indirectmeasure of the blood vessel amount) and Flow Reserve is reflected by theratio of max FL: resting FL; (c) Angiographic Score—This is measured bythe angiogram of collateral vessels. A score is determined by thepercentage of circles in an overlaying grid that with crossing opacifiedarteries divided by the total number m the rabbit thigh; (d) Capillarydensity—The number of collateral capillaries determined in lightmicroscopic sections taken from hindlimbs.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 45 Effect of Polypeptides of the Invention on Vasodilation

Since dilation of vascular endothelium is important in reducing bloodpressure, the ability of polypeptides of the invention to affect theblood pressure in spontaneously hypertensive rats (SHR) is examined.Increasing doses (0, 10, 30, 100, 300, and 900 mg/kg) of thepolypeptides of the invention are administered to 13-14 week oldspontaneously hypertensive rats (SHR). Data are expressed as themean±SEM. Statistical analysis are performed with a paired t-test andstatistical significance is defined as p<0.05 vs. the response to bufferalone.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 46 Rat Ischemic Skin Flap Model

The evaluation parameters include skin blood flow, skin temperature, andfactor VII immunohistochemistry or endothelial alkaline phosphatasereaction. Expression of polypeptides of the invention, during the skinischemia, is studied using in situ hybridization. The study in thismodel is divided into three parts as follows:

a) Ischemic skin

b) Ischemic skin wounds

c) Normal wounds

The experimental protocol includes:

a) Raising a 3×4 cm, single pedicle full-thickness random skin flap(myocutaneous flap over the lower back of the animal).

b) An excisional wounding (4-6 mm in diameter) in the ischemic skin(skin-flap).

c) Topical treatment with a polypeptide of the invention of theexcisional wounds (day 0, 1, 2, 3, 4 post-wounding) at the followingvarious dosage ranges: 1 mg to 100 mg.

d) Harvesting the wound tissues at day 3, 5, 7, 0, 14 and 21post-wounding for histological, immunohistochemical, and in situstudies.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 47 Peripheral Arterial Disease Model

Angiogenic therapy using a polypeptide of the invention is a noveltherapeutic strategy to obtain restoration of blood flow around theischemia in case of peripheral arterial diseases. The experimentalprotocol includes:

a) One side of the femoral artery is ligated to create ischemic muscleof the hindlimb, the other side of hindlimb serves as a control.

b) a polypeptide of the invention, in a dosage range of 20 mg-500 mg, isdelivered intravenously and/or intramuscularly 3 times (perhaps more)per week for 2-3 weeks.

c) The ischemic muscle tissue is collected after ligation of the femoral

artery at 1, 2, and 3 weeks for the analysis of expression of apolypeptide of the invention and histology. Biopsy is also performed onthe other side of normal muscle of the contralateral hindlimb.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 48 Ischemic Myocardial Disease Model

A polypeptide of the invention is evaluated as a potent mitogen capableof stimulating the development of collateral vessels, and restructuringnew vessels after coronary artery occlusion. Alteration of expression ofthe polypeptide is investigated in situ. The experimental protocolincludes:

a) The heart is exposed through a left-side thoracotomy in the rat.Immediately, the left coronary artery is occluded with a thin suture(6-0) and the thorax is closed.

b) a polypeptide of the invention, in a dosage range of 20 mg-500 mg, isdelivered intravenously and/or intramuscularly 3 times (perhaps more)per week for 24 weeks.

c) Thirty days after the surgery, the heart is removed andcross-sectioned for morphometric and in situ analyzes.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 49 Rat Corneal Wound Healing Model

This animal model shows the effect of a polypeptide of the invention onneovascularization. The experimental protocol includes:

a) Making a 1-1.5 mm long incision from the center of cornea into thestromal layer.

b) Inserting a spatula below the lip of the incision facing the outercorner of the eye.

c) Making a pocket (its base is 1-1.5 mm form the edge of the eye).

d) Positioning a pellet, containing 50 ng-5 ug of a polypeptide of theinvention, within the pocket.

e) Treatment with a polypeptide of the invention can also be appliedtopically to the corneal wounds in a dosage range of 20 mg-500 mg (dailytreatment for five days).

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 50 Diabetic Mouse and Glucocorticoid-Impaired Wound HealingModels

A. Diabetic db+/db+ Mouse Model.

To demonstrate that a polypeptide of the invention accelerates thehealing process, the genetically diabetic mouse model of wound healingis used. The full thickness wound healing model in the db+/db+ mouse isa well characterized, clinically relevant and reproducible model ofimpaired wound healing. Healing of the diabetic wound is dependent onformation of granulation tissue and re-epithelialization rather thancontraction (Gartner, M. H. et al., J. Surg. Res. 52:389 (1992);Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)).

The diabetic animals have many of the characteristic features observedin Type II diabetes mellitus. Homozygous (db+/db+) mice are obese incomparison to their normal heterozygous (db+/+m) littermates. Mutantdiabetic (db+/db+) mice have a single autosomal recessive mutation onchromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci. USA 77:283-293(1982)). Animals show polyphagia, polydipsia and polyuria. Mutantdiabetic mice (db+/db+) have elevated blood glucose, increased or normalinsulin levels, and suppressed cell-mediated immunity (Mandel et al., J.Immunol. 120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol.51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55 (1985)).Peripheral neuropathy, myocardial complications, and microvascularlesions, basement membrane thickening and glomerular filtrationabnormalities have been described in these animals (Norido, F. et al.,Exp. Neurol. 83(2):221-232 (1984); Robertson et al., Diabetes29(1):60-67 (1980); Giacomelli et al., Lab Invest. 40(4):460-473 (1979);Coleman, D. L., Diabetes 31 (Suppl): 1-6 (1982)). These homozygousdiabetic mice develop hyperglycemia that is resistant to insulinanalogous to human type II diabetes (Mandel et al., J. Immunol.120:1375-1377 (1978)).

The characteristics observed in these animals suggests that healing inthis model may be similar to the healing observed in human diabetes(Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).

Genetically diabetic female C57BL/KsJ (db+/db+) mice and theirnon-diabetic (db+/+m) heterozygous littermates are used in this study(Jackson Laboratories). The animals are purchased at 6 weeks of age andare 8 weeks old at the beginning of the study. Animals are individuallyhoused and received food and water ad libitum. All manipulations areperformed using aseptic techniques. The experiments are conductedaccording to the rules and guidelines of Bristol-Myers Squibb Company'sInstitutional Animal Care and Use Committee and the Guidelines for theCare and Use of Laboratory Animals.

Wounding protocol is performed according to previously reported methods(Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)).Briefly, on the day of wounding, animals are anesthetized with anintraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanoland 2-methyl-2-butanol dissolved in deionized water. The dorsal regionof the animal is shaved and the skin washed with 70% ethanol solutionand iodine. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is then created using a Keyestissue punch. Immediately following wounding, the surrounding skin isgently stretched to eliminate wound expansion. The wounds are left openfor the duration of the experiment. Application of the treatment isgiven topically for 5 consecutive days commencing on the day ofwounding. Prior to treatment, wounds are gently cleansed with sterilesaline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of surgery and at two day intervals thereafter. Wound closure isdetermined by daily measurement on days 1-5 and on day 8. Wounds aremeasured horizontally and vertically using a calibrated Jameson caliper.Wounds are considered healed if granulation tissue is no longer visibleand the wound is covered by a continuous epithelium.

A polypeptide of the invention is administered using at a rangedifferent doses, from 4 mg to 500 mg per wound per day for 8 days invehicle. Vehicle control groups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology and immunohistochemistry. Tissue specimensare placed in 10% neutral buffered formalin in tissue cassettes betweenbiopsy sponges for further processing.

Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls)are evaluated: 1) Vehicle placebo control, 2) untreated group, and 3)treated group.

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total square area of the wound.Contraction is then estimated by establishing the differences betweenthe initial wound area (day 0) and that of post treatment (day 8). Thewound area on day 1 is 64mm2, the corresponding size of the dermalpunch. Calculations are made using the following formula:[Open area on day 8]−[Open area on day 1]/ [Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embeddedblocks are sectioned perpendicular to the wound surface (5mm) and cutusing a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E)staining is performed on cross-sections of bisected wounds. Histologicexamination of the wounds are used to assess whether the healing processand the morphologic appearance of the repaired skin is altered bytreatment with a polypeptide of the invention. This assessment includedverification of the presence of cell accumulation, inflammatory cells,capillaries, fibroblasts, re-epithelialization and epidermal maturity(Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)). A calibratedlens micrometer is used by a blinded observer.

Tissue sections are also stained immunohistochemically with a polyclonalrabbit anti-human keratin antibody using ABC Elite detection system.Human skin is used as a positive tissue control while non-immune IgG isused as a negative control. Keratinocyte growth is determined byevaluating the extent of reepithelialization of the wound using acalibrated lens micrometer.

Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens isdemonstrated by using anti-PCNA antibody (1:50) with an ABC Elitedetection system. Human colon cancer can serve as a positive tissuecontrol and human brain tissue can be used as a negative tissue control.Each specimen includes a section with omission of the primary antibodyand substitution with non-immune mouse IgG. Ranking of these sections isbased on the extent of proliferation on a scale of 0-8, the lower sideof the scale reflecting slight proliferation to the higher sidereflecting intense proliferation.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

B. Steroid Impaired Rat Model

The inhibition of wound healing by steroids has been well documented invarious in vitro and in vivo systems (Wahl, Glucocorticoids and Woundhealing. In: Anti-Inflammatory Steroid Action: Basic and ClinicalAspects. 280-302 (1989); Wahiet al., J. Immunol. 115: 476-481 (1975);Werb et al., J. Exp. Med. 147:1684-1694 (1978)). Glucocorticoids retardwound healing by inhibiting angiogenesis, decreasing vascularpermeability (Ebert et al., An. Intern. Med. 37:701-705 (1952)),fibroblast proliferation, and collagen synthesis (Beck et al., GrowthFactors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797(1978)) and producing a transient reduction of circulating monocytes(Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl,“Glucocorticoids and wound healing”, In: Antiinflammatory SteroidAction: Basic and Clinical Aspects, Academic Press, New York, pp.280-302 (1989)). The systemic administration of steroids to impairedwound healing is a well establish phenomenon in rats (Beck et al.,Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61:703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In:Antiinflammatory Steroid Action: Basic and Clinical Aspects, AcademicPress, New York, pp. 280-302 (1989); Pierce et al., Proc. Natl. Acad.Sci. USA 86: 2229-2233 (1989)).

To demonstrate that a polypeptide of the invention can accelerate thehealing process, the effects of multiple topical applications of thepolypeptide on full thickness excisional skin wounds in rats in whichhealing has been impaired by the systemic administration ofmethylprednisolone is assessed.

Young adult male Sprague Dawley rats weighing 250-300 g (Charles RiverLaboratories) are used in this example. The animals are purchased at 8weeks of age and are 9 weeks old at the beginning of the study. Thehealing response of rats is impaired by the systemic administration ofmethylprednisolone (17 mg/kg/rat intramuscularly) at the time ofwounding. Animals are individually housed and received food and water adlibitum. All manipulations are performed using aseptic techniques. Thisstudy would be conducted according to the rules and guidelines ofBristol-Myers Squibb Corporations Guidelines for the Care and Use ofLaboratory Animals.

The wounding protocol is followed according to section A, above. On theday of wounding, animals are anesthetized with an intramuscularinjection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsalregion of the animal is shaved and the skin washed with 70% ethanol andiodine solutions. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is created using a Keyes tissuepunch. The wounds are left open for the duration of the experiment.Applications of the testing materials are given topically once a day for7 consecutive days commencing on the day of wounding and subsequent tomethylprednisolone administration. Prior to treatment, wounds are gentlycleansed with sterile saline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of wounding and at the end of treatment. Wound closure is determinedby daily measurement on days 1-5 and on day 8. Wounds are measuredhorizontally and vertically using a calibrated Jameson caliper. Woundsare considered healed if granulation tissue is no longer visible and thewound is covered by a continuous epithelium.

The polypeptide of the invention is administered using at a rangedifferent doses, from 4 mg to 500 mg per wound per day for 8 days invehicle. Vehicle control groups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology. Tissue specimens are placed in 10% neutralbuffered formalin in tissue cassettes between biopsy sponges for furtherprocessing.

Four groups of 10 animals each (5 with methylprednisolone and 5 withoutglucocorticoid) are evaluated: 1) Untreated group 2) Vehicle placebocontrol 3) treated groups.

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total area of the wound. Closure isthen estimated by establishing the differences between the initial woundarea (day 0) and that of post treatment (day 8). The wound area on day 1is 64 mm2, the corresponding size of the dermal punch. Calculations aremade using the following formula:[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffm embedded blocksare sectioned perpendicular to the wound surface (5 mm) and cut using anOlympus microtome. Routine hematoxylin-eosin (H&E) staining is performedon cross-sections of bisected wounds. Histologic examination of thewounds allows assessment of whether the healing process and themorphologic appearance of the repaired skin is improved by treatmentwith a polypeptide of the invention. A calibrated lens micrometer isused by a blinded observer to determine the distance of the wound gap.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background of the Invention, DetailedDescription, and Examples is hereby incorporated herein by reference.Further, the hard copy of the sequence listing submitted herewith andthe corresponding computer readable form are both incorporated herein byreference in their entireties. TABLE IV Residue Atom name x coord ycoord z coord 1 LYS12 N −27.2159 19.7201 −6.2824 2 LYS12 CA −26.697118.4591 −5.7744 3 LYS12 C −27.7349 17.3482 −5.7385 4 LYS12 O −27.410916.2063 −6.0352 5 LYS12 CB −26.1266 18.6653 −4.3594 6 LYS12 CG −24.733218.0137 −4.2714 7 LYS12 CD −24.8483 16.4855 −4.433 8 LYS12 CE −23.443915.8647 −4.5476 9 LYS12 NZ −22.7011 16.0707 −3.2959 10 LEU13 N −28.980917.682 −5.4127 11 LEU13 CA −30.064 16.6951 −5.4047 12 LEU13 C −30.253716.2158 −6.8405 13 LEU13 O −30.3861 15.0234 −7.0985 14 LEU13 CB −31.356317.3552 −4.8907 15 LEU13 CG −31.1721 17.7804 −3.421 16 LEU13 CD1−32.4543 18.4737 −2.9249 17 LEU13 CD2 −30.8835 16.5465 −2.5441 18 ALA14N −30.2416 17.1744 −7.7676 19 ALA14 CA −30.3825 16.9101 −9.195 20 ALA14C −29.2866 15.9587 −9.6613 21 ALA14 O −29.5553 14.9807 −10.3628 22 ALA14CB −30.3313 18.232 −9.9835 23 PRO15 N −28.0569 16.2571 −9.2419 24 PRO15CA −26.8772 15.4556 −9.563 25 PRO15 C −27.029 14.0783 −8.9231 26 PRO15 O−26.6765 13.0642 −9.5273 27 PRO15 CB −25.7231 16.2219 −8.8922 28 PRO15CG −26.2623 17.6337 −8.5936 29 PRO15 CD −27.7987 17.5229 −8.5821 30ARG16 N −27.58 14.0482 −7.7102 31 ARG16 CA −27.7908 12.7957 −6.9958 32ARG16 C −28.7852 11.9184 −7.7336 33 ARG16 O −28.7192 10.6897 −7.6578 34ARG16 CB −28.4328 13.1445 −5.6398 35 ARG16 CG −27.3904 13.7645 −4.691736 ARG16 CD −28.1224 14.4083 −3.4996 37 ARG16 NE −27.4893 15.6684−3.1552 38 ARG16 CZ −26.8721 15.823 −2.0196 39 ARG16 NH1 −26.319616.9665 −1.7426 40 ARG16 NH2 −26.797 14.8537 −1.1553 41 TYR17 N −29.982212.8509 −8.5832 42 TYR17 CA −30.4267 11.5765 −9.1118 43 TYR17 C −29.969711.2849 −10.5385 44 TYR17 O −30.2553 10.2132 −11.0727 45 TYR17 CB−31.971 11.5383 −9.0666 46 TYR17 CG −32.529 12.9543 −8.9375 47 TYR17 CD1−32.5301 13.8102 −10.0419 48 TYR17 CD2 −33.0158 13.3997 −7.706 49 TYR17CE1 −32.8944 15.1493 −9.8813 50 TYR17 CE2 −33.3803 14.7389 −7.5456 51TYR17 CZ −33.287 15.6204 −8.6256 52 TYR17 OH −33.5844 16.9663 −8.4507 53GLN28 N −17.3167 19.6634 −20.9577 54 GLN28 CA −16.6717 19.1545 −22.176155 GLN28 C −17.6399 18.3959 −23.1004 56 GLN28 O −17.691 18.6208 −24.308757 GLN28 CB −15.9024 20.2732 −22.9059 58 GLN28 CG −16.8571 21.4214−23.2838 59 GLN28 CD −16.0631 22.5632 −23.8481 60 GLN28 OE1 −16.252822.9259 −24.9971 61 GLN28 NE2 −15.1606 23.145 −23.0375 62 ASP29 N−17.812 18.2585 −24.2329 63 ASP29 CA −18.2937 17.1126 −24.9955 64 ASP29C −17.3081 16.7493 −26.0905 65 ASP29 O −17.1225 17.4942 −27.052 66 ASP29CB −19.7095 17.3082 −25.5761 67 ASP29 CG −20.2664 15.967 −25.9577 68ASP29 OD1 −21.0819 15.42 −25.1689 69 ASP29 OD2 −19.8983 15.4607 −27.050870 PHE30 N −16.6686 15.5986 −25.92 71 PHE30 CA −15.6883 15.1073 −26.87672 PHE30 C −16.2374 15.0541 −28.2917 73 PHE30 O −15.5848 15.4889−29.2215 74 PHE30 CB −15.0913 13.7425 −26.4865 75 PHE30 CG −13.931213.4564 −27.4363 76 PHE30 CD1 −12.8189 14.302 −27.4553 77 PHE30 CD2−13.9836 12.3569 −28.2963 78 PHE30 CE1 −11.7856 14.0734 −28.367 79 PHE30CE2 −12.9192 12.0914 −29.1615 80 PHE30 CZ −11.8222 12.9557 −29.2041 81GLU31 N −17.4227 14.4765 −28.4391 82 GLU31 CA −18.0695 14.3384 −29.74 83GLU31 C −18.4961 15.6958 −30.2978 84 GLU31 O −18.1746 16.0281 −31.436685 GLU31 CB −19.3248 13.4596 −29.5707 86 GLU31 CG −18.9969 12.1952−28.7512 87 GLU31 CD −19.1929 12.4724 −27.2876 88 GLU31 OE1 −20.372812.5627 −26.8547 89 GLU31 OE2 −18.1684 12.5927 −26.5636 90 ALA32 N−19.1724 16.4914 −29.4728 91 ALA32 CA −19.6256 17.817 −29.8767 92 ALA32C −18.4792 18.7175 −30.3487 93 ALA32 O −18.5771 19.3496 −31.4051 94ALA32 CB −20.3076 18.4749 −28.6631 95 LEU33 N −17.3954 18.7607 −29.575196 LEU33 CA −16.2231 19.5772 −29.9031 97 LEU33 C −15.4331 19.0228−31.0884 98 LEU33 O −14.8897 19.7818 −31.891 99 LEU33 CB −15.301119.5611 −28.6684 100 LEU33 CG −16.0154 20.2097 −27.4667 101 LEU33 CD1−15.182 19.9944 −26.1896 102 LEU33 CD2 −16.1953 21.7178 −27.7216 103LEU34 N −15.3571 17.6969 −31.1802 104 LEU34 CA −14.6678 17.0334 −32.2855105 LEU34 C −15.4723 17.3381 −33.5408 106 LEU34 O −14.9271 17.7085−34.5669 107 LEU34 CB −14.7381 15.5166 −32.0247 108 LEU34 CG −13.430814.8282 −32.4594 109 LEU34 CD1 −13.4802 13.3423 −32.0608 110 LEU34 CD2−13.2453 14.9435 −33.9839 111 ALA35 N −16.7854 17.2046 −33.4234 112ALA35 CA −17.7074 17.4638 −34.5157 113 ALA35 C −17.5894 18.9123 −34.9803114 ALA35 O −17.5165 19.1899 −36.1781 115 ALA35 CB −19.1407 17.2174−34.0109 116 GLU36 N −17.5819 19.8209 −34.0104 117 GLU36 CA −17.461621.2591 −34.2319 118 GLU36 C −16.2171 21.5709 −35.0692 119 GLU36 O−16.2857 22.2732 −36.0777 120 GLU36 CB −17.3366 21.903 −32.8349 121GLU36 CG −16.8291 23.3574 −32.9143 122 GLU36 CD −15.3265 23.3815−32.8896 123 GLU36 OE1 −14.7352 22.8224 −31.927 124 GLU36 OE2 −14.731623.9552 −33.8408 125 CYS37 N −15.0851 21.0145 −34.6518 126 CYS37 CA−13.8228 21.2122 −35.3467 127 CYS37 C −13.8265 20.4262 −36.6526 128CYS37 O −13.2082 20.8268 −37.6325 129 CYS37 CB −12.7031 20.6447 −34.4549130 CYS37 SG −11.7988 22.0477 −33.7396 131 LEU38 N −14.5137 19.2872−36.6354 132 LEU38 CA −14.6365 18.4176 −37.7991 133 LEU38 C −15.242719.2608 −38.8935 134 LEU38 O −14.7294 19.3257 −40.0108 135 LEU38 CB−15.5583 17.2359 −37.4444 136 LEU38 CG −14.8353 15.9028 −37.7188 137LEU38 CD1 −13.5226 15.8275 −36.9155 138 LEU38 CD2 −15.7503 14.7352−37.3062 139 ARG39 N −16.3056 19.9654 −38.5243 140 ARG39 CA −17.029720.8292 −39.4381 141 ARG39 C −16.244 22.0579 −39.8902 142 ARG39 O−15.9049 22.1629 −41.0629 143 ARG39 CB −18.3186 21.316 −38.7483 144ARG39 CG −19.1868 20.1166 −38.3268 145 ARG39 CD −20.103 20.5459 −37.1664146 ARG39 NE −20.3618 19.4053 −36.3068 147 ARG39 CZ −21.5716 18.9628−36.1218 148 ARG39 NH1 −21.7652 17.9388 −35.3449 149 ARG39 NH2 −22.591319.5263 −36.7005 150 ASN40 N −15.8973 22.9463 −38.9598 151 ASN40 CA−15.158 24.169 −39.2938 152 ASN40 C −13.7759 23.9349 −39.8974 153 ASN40O −13.082 24.8845 −40.2596 154 ASN40 CB −14.9873 25.0138 −38.0155 155ASN40 CG −16.3169 25.3188 −37.3873 156 ASN40 OD1 −16.5252 25.0099−36.2256 157 ASN40 ND2 −17.2341 25.9335 −38.1556 158 GLY41 N −13.389322.667 −40.0176 159 GLY41 CA −12.0967 22.3184 −40.5824 160 GLY41 C−10.9452 22.9275 −39.8082 161 GLY41 O −10.0967 23.6122 −40.3791 162CYS42 N −10.9167 22.6693 −38.5057 163 CYS42 CA −9.8803 23.2064 −37.6375164 CYS42 C −9.4616 22.2276 −36.5439 165 CYS42 O −9.9692 21.1126−36.4619 166 CYS42 CB −10.4265 24.4707 −36.9437 167 CYS42 SG −10.75325.7585 −38.1837 168 LEU43 N −8.5193 22.6577 −35.7118 169 LEU43 CA−8.0132 21.8337 −34.6213 170 LEU43 C −8.4703 22.3473 −33.2651 171 LEU43O −8.5778 23.5611 −33.0479 172 LEU43 CB −6.4773 21.7117 −34.6571 173LEU43 CG −5.9576 21.4349 −36.0819 174 LEU43 CD1 −4.4199 21.3664 −36.0489175 LEU43 CD2 −6.5092 20.0994 −36.615 176 PHE44 N −8.6955 21.4162−32.3455 177 PHE44 CA −9.1464 21.7627 −31.0043 178 PHE44 C −8.088322.4337 −30.1214 179 PHE44 O −6.9495 21.9754 −29.9899 180 PHE44 CB−9.8202 20.5294 −30.3676 181 PHE44 CG −9.882 20.6174 −28.8472 182 PHE44CD1 −10.7478 21.5258 −28.233 183 PHE44 CD2 −9.0725 19.7821 −28.073 184PHE44 CE1 −10.8371 21.5679 −26.8396 185 PHE44 CE2 −9.1608 19.8264−26.6796 186 PHE44 CZ −10.0598 20.7022 −26.0661 187 GLU45 N −8.481123.5795 −29.5902 188 GLU45 CA −7.6634 24.3474 −28.6926 189 GLU45 C−8.5174 24.3954 −27.4403 190 GLU45 O −9.5537 25.0596 −27.3985 191 GLU45CB −7.4462 25.7576 −29.2734 192 GLU45 CG −6.7926 25.6599 −30.6663 193GLU45 CD −5.4031 25.1005 −30.5591 194 GLU45 OE1 −4.5005 25.842 −30.0872195 GLU45 OE2 −5.2083 23.9213 −30.9566 196 ASP46 N −8.1279 23.5759−26.4713 197 ASP46 CA −8.8271 23.4565 −25.2064 198 ASP46 C −8.9745 24.79−24.4679 199 ASP46 O −7.9792 25.4257 −24.1015 200 ASP46 CB −7.997322.5011 −24.3257 201 ASP46 CG −8.8587 21.8017 −23.3145 202 ASP46 OD1−9.4405 22.4985 −22.4406 203 ASP46 OD2 −8.9499 20.5471 −23.39 204 THR47N −10.2319 25.236 −24.2636 205 THR47 CA −10.5788 26.4827 −23.5731 206THR47 C −10.5448 26.3291 −22.0414 207 THR47 O −10.6702 27.3036 −21.297208 THR47 CB −11.991 26.9051 −24.0256 209 THR47 OG1 −12.0685 26.8763−25.4538 210 THR47 CG2 −12.2993 28.3333 −23.5373 211 SER48 N −10.356225.0947 −21.5841 212 SER48 CA −10.3202 24.7963 −20.1625 213 SER48 C−8.9327 24.3765 −19.7007 214 SER48 O −8.7044 24.1661 −18.5061 215 SER48CB −11.3054 23.6448 −19.8871 216 SER48 OG −12.6213 24.0291 −20.2945 217PHE49 N −8.0123 24.2454 −20.6548 218 PHE49 CA −6.6435 23.8505 −20.3588219 PHE49 C −5.7089 24.3915 −21.4418 220 PHE49 O −5.1154 23.6305−22.2142 221 PHE49 CB −6.5801 22.3116 −20.2651 222 PHE49 CG −5.329821.8633 −19.5126 223 PHE49 CD1 −5.3312 21.8194 −18.1156 224 PHE49 CD2−4.1833 21.4888 −20.2187 225 PHE49 CE1 −4.1956 21.3837 −17.4269 226PHE49 CE2 −3.047 21.0549 −19.5305 227 PHE49 CZ −3.0541 20.9974 −18.1343228 PRO50 N −5.5643 25.7267 −21.5041 229 PRO50 CA −4.7293 26.4558−22.4561 230 PRO50 C −3.2687 26.0165 −22.478 231 PRO50 O −2.7439 25.4808−21.4869 232 PRO50 CB −4.8313 27.9279 −22.0172 233 PRO50 CG −5.816527.9803 −20.831 234 PRO50 CD −6.2821 26.5397 −20.5405 235 ALA51 N−2.6121 26.2739 −23.6058 236 ALA51 CA −1.2098 25.9377 −23.7851 237 ALA51C −0.3497 26.9614 −23.0391 238 ALA51 O 0.7223 27.3612 −23.5021 239 ALA51CB −0.8481 25.8622 −25.281 240 THR52 N −0.8405 27.3848 −21.8767 241THR52 CA −0.1559 28.3625 −21.0525 242 THR52 C 0.6958 27.7065 −19.9669243 THR52 O 0.709 26.4813 −19.8398 244 THR52 CB −1.2136 29.2515 −20.369245 THR52 OG1 −2.0784 28.4487 −19.5588 246 THR52 CG2 −2.0467 29.9875−21.4353 247 LEU53 N 1.5163 28.5131 −19.2586 248 LEU53 CA 2.3863 28.037−18.1786 249 LEU53 C 1.5758 27.5585 −16.9718 250 LEU53 O 2.0681 26.7947−16.1515 251 LEU53 CB 3.3336 29.1723 −17.7501 252 LEU53 CG 4.609929.1306 −18.6121 253 LEU53 CD1 5.5137 30.3224 −18.247 254 LEU53 CD25.3734 27.8177 −18.3543 255 SER54 N 0.3319 28.0127 −16.8819 256 SER54 CA−0.569 27.6427 −15.7938 257 SER54 C −0.9496 26.1564 −15.8412 258 SER54 O−1.2905 25.5583 −14.8192 259 SER54 CB −1.8575 28.4696 −15.9596 260 SER54OG −1.5491 29.864 −15.8839 261 SER55 N −0.9164 25.584 −17.0444 262 SER55CA −1.2204 24.1714 −17.2571 263 SER55 C −0.0414 23.3364 −16.7588 264SER55 O −0.2092 22.2061 −16.2832 265 SER55 CB −1.469 23.9085 −18.7566266 SER55 OG −0.2429 23.9254 −19.4941 267 ILE56 N 1.1563 23.8991−16.9025 268 ILE56 CA 2.3905 23.2585 −16.4678 269 ILE56 C 2.5322 23.3656−14.9373 270 ILE56 O 2.886 22.3901 −14.2855 271 ILE56 CB 3.5575 23.9946−17.1549 272 ILE56 CG1 3.3514 23.9704 −18.6829 273 ILE56 CG2 4.891923.3115 −16.798 274 ILE56 CD1 4.3716 24.8964 −19.3715 275 GLY57 N 2.264124.5079 −14.3713 276 GLY57 CA 2.3598 24.7298 −12.9266 277 GLY57 C 3.799624.4235 −12.4989 278 GLY57 O 4.2274 23.2821 −12.5859 279 LEU65 N 12.311525.9569 −15.7051 280 LEU65 CA 12.1314 24.6761 −16.4042 281 LEU65 C11.0916 24.8704 −17.538 282 LEU65 O 11.2794 24.3854 −18.6495 283 LEU65CB 11.5248 23.6404 −15.4356 284 LEU65 CG 12.5547 23.1647 −14.3945 285LEU65 CD1 11.8521 22.277 −13.351 286 LEU65 CD2 13.6664 22.3566 −15.0896287 PRO66 N 10.0696 25.653 −17.3149 288 PRO66 CA 9.0282 25.8331 −18.322289 PRO66 C 9.2286 27.0866 −19.1893 290 PRO66 O 8.2609 27.7518 −19.5641291 PRO66 CB 7.7834 26.0207 −17.4333 292 PRO66 CG 8.2941 26.5192−16.0641 293 PRO66 CD 9.8033 26.2118 −16.0068 294 PRO67 N 10.484227.3788 −19.5276 295 PRO67 CA 10.8245 28.5473 −20.3404 296 PRO67 C11.2929 28.1703 −21.7447 297 PRO67 O 12.161 27.3217 −21.9075 298 PRO67CB 12.0302 29.1474 −19.5952 299 PRO67 CG 12.6726 27.9643 −18.8471 300PRO67 CD 11.6812 26.7941 −18.9858 301 ARG68 N 10.701 28.7991 −22.754 302ARG68 CA 11.0793 28.5286 −24.135 303 ARG68 C 10.7253 27.1529 −24.6599304 ARG68 O 11.4609 26.5524 −25.4496 305 ARG68 CB 12.5578 28.8764−24.4017 306 ARG68 CG 12.8181 30.3504 −24.0425 307 ARG68 CD 14.323730.6448 −24.1658 308 ARG68 NE 14.5854 31.9972 −23.709 309 ARG68 CZ15.264 32.2199 −22.6207 310 ARG68 NH1 15.4798 33.445 −22.2438 311 ARG68NH2 15.7293 31.2393 −21.9035 312 LEU69 N 9.5924 26.6432 −24.2168 313LEU69 CA 9.1505 25.3393 −24.6592 314 LEU69 C 7.8526 25.4596 −25.456 315LEU69 O 6.9058 26.1202 −25.0331 316 LEU69 CB 8.9084 24.3992 −23.457 317LEU69 CG 8.7632 25.1657 −22.1248 318 LEU69 CD1 7.4743 26.0088 −22.1242319 LEU69 CD2 8.7105 24.1644 −20.9568 320 GLN70 N 7.8405 24.8246−26.6228 321 GLN70 CA 6.7115 24.8565 −27.5489 322 GLN70 C 5.7536 23.6717−27.4633 323 GLN70 O 6.1667 22.5133 −27.4887 324 GLN70 CB 7.2115 24.9668−29.0024 325 GLN70 CG 8.2553 26.0927 −29.1291 326 GLN70 CD 9.624525.5437 −28.8497 327 GLN70 OE1 10.2487 25.9357 −27.8779 328 GLN70 NE210.105 24.6265 −29.7079 329 TRP71 N 4.465 23.9829 −27.3854 330 TRP71 CA3.4258 22.9642 −27.3331 331 TRP71 C 3.1746 22.4423 −28.7393 332 TRP71 O2.4693 23.0698 −29.5419 333 TRP71 CB 2.1377 23.5984 −26.7739 334 TRP71CG 2.312 23.8643 −25.3078 335 TRP71 CD1 2.8263 24.9684 −24.7439 336TRP71 CD2 1.94 22.9265 −24.192 337 TRP71 NE1 2.8321 24.8419 −23.4397 338TRP71 CE2 2.3239 23.6463 −23.0764 339 TRP71 CE3 1.3639 21.6594 −24.1194340 TRP71 CZ2 2.1738 23.1341 −21.788 341 TRP71 CZ3 1.2152 21.1334−22.8307 342 TRP71 CH2 1.6176 21.8522 −21.6955 343 LYS72 N 3.800621.3124 −29.0428 344 LYS72 CA 3.6586 20.6789 −30.3357 345 LYS72 C 2.695119.49 −30.1769 346 LYS72 O 2.6561 18.7499 −29.2878 347 LYS72 CB 5.038420.2965 −30.9014 348 LYS72 CG 5.9482 21.5389 −30.8451 349 LYS72 CD7.3908 21.1728 −31.2361 350 LYS72 CE 8.3114 22.3529 −30.8736 351 LYS72NZ 9.384 22.4688 −31.8714 352 ARG73 N 1.887 19.3481 −31.3241 353 ARG73CA 0.9543 18.2391 −31.4196 354 ARG73 C 1.7324 17.134 −32.1228 355 ARG73O 2.7467 17.4047 −32.7786 356 ARG73 CB −0.2282 18.6367 −32.3243 357ARG73 CG −1.0143 19.8161 −31.7239 358 ARG73 CD −2.4537 19.344 −31.4564359 ARG73 NE −3.4166 20.2295 −32.081 360 ARG73 CZ −4.5688 20.4563−31.5206 361 ARG73 NH1 −4.888 19.8999 −30.3895 362 ARG73 NH2 −5.415721.2509 −32.1024 363 PRO74 N 1.299 15.8673 −31.9654 364 PRO74 CA 1.948914.7052 −32.5823 365 PRO74 C 2.1161 14.8143 −34.1023 366 PRO74 O 2.974114.1518 −34.6932 367 PRO74 CB 0.9333 13.5833 −32.2893 368 PRO74 CG−0.0003 14.0863 −31.1698 369 PRO74 CD 0.1523 15.6169 −31.1164 370 PRO75N 1.2926 15.6575 −34.7186 371 PRO75 CA 1.316 15.8916 −36.1551 372 PRO75C 2.6997 16.3246 −36.5983 373 PRO75 O 3.2238 15.8412 −37.6031 374 PRO75CB 0.2915 17.0171 −36.3851 375 PRO75 CG −0.6163 17.027 −35.1413 376PRO75 CD 0.1047 16.187 −34.0712 377 GLU76 N 3.2625 17.3134 −35.8124 378GLU76 CA 4.4344 18.0945 −36.1982 379 GLU76 C 5.7322 17.2973 −35.9839 380GLU76 O 6.7912 17.6086 −36.527 381 GLU76 CB 4.5004 19.329 −35.2796 382GLU76 CG 3.3103 20.2616 −35.5694 383 GLU76 CD 2.2824 20.1052 −34.4879384 GLU76 OE1 2.3162 20.9111 −33.5203 385 GLU76 OE2 1.4303 19.1867−34.6128 386 LEU77 N 5.6511 16.3624 −34.9705 387 LEU77 CA 6.8391 15.8446−34.2955 388 LEU77 C 7.4537 14.6814 −35.0966 389 LEU77 O 8.6507 14.4037−34.9876 390 LEU77 CB 6.3855 15.2602 −32.9414 391 LEU77 CG 5.316116.1448 −32.2704 392 LEU77 CD1 4.7594 15.4336 −31.0233 393 LEU77 CD25.9284 17.4984 −31.8652 394 HIS78 N 6.5733 13.885 −35.8006 395 HIS78 CA7.0516 12.7057 −36.5296 396 HIS78 C 6.221 12.4993 −37.8079 397 HIS78 O5.1972 13.1305 −38.0482 398 HIS78 CB 6.9335 11.4315 −35.6679 399 HIS78CG 7.2747 11.6999 −34.231 400 HIS78 ND1 6.394 12.1963 −33.3935 401 HIS78CD2 8.4737 11.4727 −33.6617 402 HIS78 CE1 6.9793 12.313 −32.244 403HIS78 NE2 8.1679 11.9184 −32.3293 404 SER79 N 6.744 11.5449 −38.6654 405SER79 CA 6.2259 11.3421 −40.0167 406 SER79 C 4.9636 10.4715 −40.0374 407SER79 O 4.1412 10.5656 −40.9488 408 SER79 CB 7.3217 10.6505 −40.8505 409SER79 OG 7.6769 9.3977 −40.2577 410 ASN80 N 4.9115 9.4953 −39.0614 411ASN80 CA 3.7097 8.6832 −38.8418 412 ASN80 C 3.3877 8.7954 −37.3409 413ASN80 O 3.9182 8.0475 −36.5135 414 ASN80 CB 4.0308 7.2212 −39.216 415ASN80 CG 2.8922 6.2781 −38.9316 416 ASN80 OD1 1.8258 6.6843 −38.4983 417ASN80 ND2 3.1233 4.9769 −39.1805 418 PRO81 N 2.5675 9.8295 −36.9434 419PRO81 CA 2.0432 9.8923 −35.5806 420 PRO81 C 0.9728 8.7992 −35.4421 421PRO81 O −0.0481 8.7744 −36.1332 422 PRO81 CB 1.3513 11.2709 −35.5758 423PRO81 CG 1.275 11.7676 −37.0367 424 PRO81 CD 2.1119 10.8087 −37.9067 425GLN82 N 1.2765 7.8106 −34.525 426 GLN82 CA 0.2983 6.7865 −34.207 427GLN82 C −0.3517 7.1645 −32.8705 428 GLN82 O 0.2082 7.817 −31.9873 429GLN82 CB 1.0091 5.4307 −34.0417 430 GLN82 CG 1.6042 5.0014 −35.3964 431GLN82 CD 0.583 4.3049 −36.2521 432 GLN82 OE1 −0.5485 4.1029 −35.8418 433GLN82 NE2 0.9893 3.9225 −37.4754 434 PHE83 N −1.6335 6.6481 −32.7332 435PHE83 CA −2.2914 6.7368 −31.4406 436 PHE83 C −1.7335 5.594 −30.5892 437PHE83 O −1.0996 5.8522 −29.5679 438 PHE83 CB −3.8224 6.6601 −31.5886 439PHE83 CG −4.4571 7.7012 −30.672 440 PHE83 CD1 −4.1099 9.0492 −30.7975441 PHE83 CD2 −5.3827 7.3067 −29.7024 442 PHE83 CE1 −4.6423 9.9939−29.9166 443 PHE83 CE2 −5.929 8.2541 −28.8324 444 PHE83 CZ −5.55029.5954 −28.9318 445 ASP92 N 8.7138 1.3421 −24.7856 446 ASP92 CA 9.93332.1258 −24.6143 447 ASP92 C 11.0305 1.4805 −25.5119 448 ASP92 O 10.75460.699 −26.4246 449 ASP92 CB 10.407 2.1775 −23.1449 450 ASP92 CG 9.72483.2612 −22.3582 451 ASP92 OD1 9.2374 4.2466 −22.9756 452 ASP92 OD29.6647 3.1203 −21.1075 453 LEU93 N 12.3299 1.9183 −25.302 454 LEU93 CA13.4513 1.2276 −25.9597 455 LEU93 C 13.9838 0.2162 −24.9175 456 LEU93 O13.6738 −0.9711 −24.9345 457 LEU93 CB 14.548 2.2237 −26.3839 458 LEU93CG 13.9455 3.3288 −27.2731 459 LEU93 CD1 15.0357 4.3611 −27.6145 460LEU93 CD2 13.3912 2.7184 −28.5745 461 CYS94 N 14.7737 0.7976 −23.9419462 CYS94 CA 15.3553 0.0678 −22.8027 463 CYS94 C 15.5177 1.1607 −21.7285464 CYS94 O 15.0841 1.0768 −20.5819 465 CYS94 CB 16.7457 −0.4768−23.1815 466 CYS94 SG 16.5289 −1.9599 −24.2076 467 GLN95 N 16.19112.2917 −22.1814 468 GLN95 CA 16.3642 3.4819 −21.3572 469 GLN95 C 15.03234.2548 −21.3495 470 GLN95 O 14.8373 5.2658 −22.0181 471 GLN95 CB 17.4874.3453 −21.962 472 GLN95 CG 18.8381 3.6285 −21.7829 473 GLN95 CD 19.94434.4988 −22.304 474 GLN95 OE1 20.6087 4.1307 −23.2586 475 GLN95 NE220.1536 5.6691 −21.6745 476 GLY96 N 14.0855 3.7102 −20.5023 477 GLY96 CA12.7456 4.2719 −20.3779 478 GLY96 C 12.6237 5.1896 −19.1601 479 GLY96 O11.5433 5.522 −18.6759 480 ILE97 N 13.8142 5.7522 −18.7335 481 ILE97 CA13.9329 6.3569 −17.4039 482 ILE97 C 13.1112 7.6447 −17.1971 483 ILE97 O12.9717 8.1418 −16.0811 484 ILE97 CB 15.428 6.6321 −17.1406 485 ILE97CG1 16.2594 5.3785 −17.4804 486 ILE97 CG2 15.6462 7.0035 −15.6609 487ILE97 CD1 17.7616 5.7174 −17.456 488 VAL98 N 12.6429 8.2331 −18.3549 489VAL98 CA 11.7495 9.3858 −18.3377 490 VAL98 C 10.252 8.9878 −18.3753 491VAL98 O 9.3646 9.8424 −18.3641 492 VAL98 CB 12.0704 10.3188 −19.5223 493VAL98 CG1 13.5084 10.8545 −19.3896 494 VAL98 CG2 11.9206 9.5627 −20.8569495 GLY99 N 9.9718 7.6392 −18.4239 496 GLY99 CA 8.6184 7.1278 −18.3169497 GLY99 C 8.2272 6.8629 −16.8609 498 GLY99 O 9.0457 6.6416 −15.9726499 ASP100 N 6.8553 6.8641 −16.6641 500 ASP100 CA 6.2623 6.5337 −15.3641501 ASP100 C 5.831 5.0446 −15.3993 502 ASP100 O 5.6976 4.4108 −16.4423503 ASP100 CB 5.0095 7.4013 −15.1404 504 ASP100 CG 4.7068 7.4901−13.6731 505 ASP100 OD1 5.1626 8.4764 −13.0357 506 ASP100 OD2 4.00116.5834 −13.1557 507 CYS101 N 5.6063 4.4998 −14.1533 508 CYS101 CA 5.51673.0637 −13.8994 509 CYS101 C 4.1114 2.4834 −14.084 510 CYS101 O 3.93751.2843 −14.319 511 CYS101 CB 6.0579 2.7259 −12.4996 512 CYS101 SG 6.82611.0845 −12.6355 513 TRP102 N 3.0511 3.3388 −13.8879 514 TRP102 CA 1.67442.8555 −14.0637 515 TRP102 C 1.3911 2.5082 −15.5374 516 TRP102 O 0.5211.7013 −15.8714 517 TRP102 CB 0.7186 3.986 −13.6263 518 TRP102 CG 0.72175.1136 −14.6207 519 TRP102 CD1 1.4421 6.2439 −14.5577 520 TRP102 CD2−0.0869 5.1729 −15.889 521 TRP102 NE1 1.1984 6.9888 −15.6076 522 TRP102CE2 0.3029 6.3884 −16.4185 523 TRP102 CE3 −1.0105 4.334 −16.5113 524TRP102 CZ2 −0.2065 6.8568 −17.6295 525 TRP102 CZ3 −1.5256 4.7953−17.7289 526 TRP102 CH2 −1.1332 6.0272 −18.2731 527 PHE103 N 2.17993.1817 −16.4499 528 PHE103 CA 1.97 3.0654 −17.8948 529 PHE103 C 2.261.6029 −18.2845 530 PHE103 O 1.6619 1.0124 −19.1813 531 PHE103 CB 2.96864.0053 −18.5934 532 PHE103 CG 2.3833 4.5108 −19.9084 533 PHE103 CD10.9999 4.5431 −20.1068 534 PHE103 CD2 3.2434 4.9464 −20.9194 535 PHE103CE1 0.4757 4.9946 −21.3197 536 PHE103 CE2 2.7199 5.416 −22.1263 537PHE103 CZ 1.3397 5.3971 −22.3407 538 LEU104 N 3.3014 1.0417 −17.5789 539LEU104 CA 3.8472 −0.2778 −17.8623 540 LEU104 C 2.8276 −1.3489 −17.424541 LEU104 O 2.6573 −2.3938 −18.0553 542 LEU104 CB 5.1372 −0.4506−17.0364 543 LEU104 CG 6.3276 0.2252 −17.7462 544 LEU104 CD1 6.11771.7499 −17.8154 545 LEU104 CD2 7.6261 −0.0697 −16.973 546 ALA105 N2.1932 −1.0749 −16.2233 547 ALA105 CA 1.1276 −1.9355 −15.7244 548 ALA105C −0.1106 −1.8301 −16.6245 549 ALA105 O −0.8567 −2.7933 −16.8042 550ALA105 CB 0.7518 −1.4414 −14.3154 551 ALA106 N −0.3581 −0.5791 −17.1656552 ALA106 CA −1.487 −0.3921 −18.0657 553 ALA106 C −1.22 −1.1369 −19.392554 ALA106 O −2.1309 −1.6826 −20.017 555 ALA106 CB −1.5879 1.1155−18.3643 556 LEU107 N 0.0829 −1.0988 −19.8597 557 LEU107 CA 0.4617−1.7496 −21.1241 558 LEU107 C 0.418 −3.2763 −20.9538 559 LEU107 O 0.1156−4.0209 −21.8892 560 LEU107 CB 1.8699 −1.2468 −21.4836 561 LEU107 CG1.7736 0.2519 −21.8284 562 LEU107 CD1 3.1721 0.8904 −21.8436 563 LEU107CD2 1.0896 0.4491 −23.1921 564 GLN108 N 0.7506 −3.7767 −19.7065 565GLN108 CA 0.7222 −5.2282 −19.4859 566 GLN108 C −0.7099 −5.7747 −19.7101567 GLN108 O −0.933 −6.9481 −20.0032 568 GLN108 CB 1.1255 −5.4721−18.0208 569 GLN108 CG 2.6571 −5.3768 −17.8879 570 GLN108 CD 3.0337−5.1535 −16.4519 571 GLN108 OE1 3.7327 −4.2021 −16.1437 572 GLN108 NE22.5673 −6.0397 −15.5542 573 ALA109 N −1.6921 −4.8226 −19.5244 574 ALA109CA −3.1166 −5.0242 −19.7711 575 ALA109 C −3.5236 −4.8583 −21.2595 576ALA109 O −4.7013 −4.7503 −21.6008 577 ALA109 CB −3.9504 −4.0865 −18.8771578 LEU110 N −2.5076 −5.0135 −22.1825 579 LEU110 CA −2.7165 −5.3006−23.6053 580 LEU110 C −2.2962 −6.7648 −23.8919 581 LEU110 O −1.8484−7.1455 −24.9707 582 LEU110 CB −1.9913 −4.2966 −24.5207 583 LEU110 CG−2.7136 −2.9386 −24.4503 584 LEU110 CD1 −2.133 −2.0943 −23.2995 585LEU110 CD2 −2.5442 −2.1967 −25.7885 586 ALA111 N −2.6269 −7.6359−22.8693 587 ALA111 CA −2.5563 −9.0971 −23.007 588 ALA111 C −3.8628−9.5213 −23.7149 589 ALA111 O −4.7955 −8.7411 −23.9273 590 ALA111 CB−2.5057 −9.7403 −21.6089 591 LEU112 N −3.9772 −10.8455 −24.0885 592LEU112 CA −5.1466 −11.3563 −24.8259 593 LEU112 C −6.3949 −11.5635−23.9092 594 LEU112 O −7.2128 −12.4664 −24.0847 595 LEU112 CB −4.7856−12.6517 −25.5768 596 LEU112 CG −4.4419 −12.3208 −27.0409 597 LEU112 CD1−3.8983 −13.5827 −27.7356 598 LEU112 CD2 −5.7085 −11.8411 −27.7752 599ILE116 N −8.104 −3.5179 −19.3071 600 ILE116 CA −8.2759 −2.3901 −18.3846601 ILE116 C −7.8945 −1.0951 −19.1202 602 ILE116 O −8.4238 −0.0119−18.8715 603 ILE116 CB −7.2846 −2.5885 −17.2211 604 ILE116 CG1 −7.3947−4.0199 −16.6603 605 ILE116 CG2 −7.5752 −1.5673 −16.1043 606 ILE116 CD1−6.1486 −4.3382 −15.8139 607 LEU117 N −6.907 −1.2245 −20.0738 608 LEU117CA −6.3848 −0.0669 −20.8005 609 LEU117 C −7.5499 0.6023 −21.5521 610LEU117 O −7.6652 1.8281 −21.6358 611 LEU117 CB −5.3551 −0.6028 −21.8165612 LEU117 CG −4.3222 0.4745 −22.206 613 LEU117 CD1 −4.997 1.6243−22.9783 614 LEU117 CD2 −3.6102 1.0177 −20.9523 615 SER118 N −8.4382−0.2753 −22.1503 616 SER118 CA −9.527 0.208 −22.9924 617 SER118 C−10.6426 0.9255 −22.2129 618 SER118 O −11.551 1.5242 −22.7887 619 SER118CB −10.1172 −0.9766 −23.7808 620 SER118 OG −10.6208 −1.9642 −22.8771 621ARG119 N −10.569 0.8227 −20.8375 622 ARG119 CA −11.4775 1.5553 −19.9688623 ARG119 C −11.0002 3.0022 −19.7933 624 ARG119 O −11.7956 3.914−19.5696 625 ARG119 CB −11.4061 0.886 −18.5811 626 ARG119 CG −11.688−0.6251 −18.6941 627 ARG119 CD −11.0396 −1.3648 −17.5085 628 ARG119 NE−11.8994 −1.2975 −16.3406 629 ARG119 CZ −11.4917 −0.7479 −15.2328 630ARG119 NH1 −10.3021 −0.2312 −15.1306 631 ARG119 NH2 −12.2908 −0.7158−14.2078 632 VAL120 N −9.631 3.1517 −19.7311 633 VAL120 CA −8.98084.4338 −19.4573 634 VAL120 C −8.8841 5.2185 −20.7772 635 VAL120 O−9.0856 6.4347 −20.8279 636 VAL120 CB −7.5615 4.1665 −18.9181 637 VAL120CG1 −6.8398 5.5013 −18.6496 638 VAL120 CG2 −7.6483 3.366 −17.6051 639VAL121 N −8.408 4.4728 −21.8391 640 VAL121 CA −8.1138 5.031 −23.1509 641VAL121 C −9.1408 4.4303 −24.128 642 VAL121 O −9.0317 3.276 −24.5517 643VAL121 CB −6.6705 4.6559 −23.5399 644 VAL121 CG1 −6.3261 5.265 −24.9123645 VAL121 CG2 −5.6994 5.2154 −22.4827 646 PRO122 N −10.1876 5.2355−24.526 647 PRO122 CA −11.1232 4.7921 −25.5543 648 PRO122 C −10.33694.6489 −26.8664 649 PRO122 O −9.7674 5.5885 −27.4207 650 PRO122 CB−12.0732 6.0025 −25.6621 651 PRO122 CG −11.4638 7.1599 −24.8396 652PRO122 CD −10.3163 6.5757 −23.9929 653 LEU123 N −10.2932 3.3366 −27.3276654 LEU123 CA −9.3386 2.9467 −28.3737 655 LEU123 C −9.728 3.5064−29.7541 656 LEU123 O −8.9297 3.5627 −30.6897 657 LEU123 CB −9.26191.4084 −28.4148 658 LEU123 CG −8.9489 0.858 −27.008 659 LEU123 CD1−8.9634 −0.6812 −27.0383 660 LEU123 CD2 −7.5671 1.3497 −26.5353 661ASN124 N −11.0556 3.8432 −29.8956 662 ASN124 CA −11.6259 4.3803 −31.1327663 ASN124 C −11.1889 5.8395 −31.3775 664 ASN124 O −11.9811 6.7626−31.546 665 ASN124 CB −13.1634 4.2757 −31.0468 666 ASN124 CG −13.70085.0409 −29.8683 667 ASN124 OD1 −14.4125 6.0144 −30.0524 668 ASN124 ND2−13.3664 4.6068 −28.6394 669 GLN125 N −9.822 6.007 −31.4634 670 GLN125CA −9.1814 7.3052 −31.6147 671 GLN125 C −7.9466 7.1256 −32.5074 672GLN125 O −7.3304 6.0632 −32.5861 673 GLN125 CB −8.7584 7.8764 −30.2481674 GLN125 CG −10.0007 8.1186 −29.3703 675 GLN125 CD −9.6273 8.9664−28.1894 676 GLN125 OE1 −10.1086 10.0807 −28.068 677 GLN125 NE2 −8.76168.4413 −27.3035 678 SER126 N −7.5777 8.2638 −33.2006 679 SER126 CA−6.5795 8.2017 −34.263 680 SER126 C −6.11 9.618 −34.6132 681 SER126 O−6.7578 10.6218 −34.3314 682 SER126 CB −7.1575 7.5132 −35.5138 683SER126 OG −6.1045 7.2436 −36.4424 684 PHE127 N −4.9112 9.6319 −35.3035685 PHE127 CA −4.4128 10.8268 −35.9869 686 PHE127 C −4.7745 10.8026−37.4801 687 PHE127 O −4.6802 11.8116 −38.1782 688 PHE127 CB −2.878210.8614 −35.8505 689 PHE127 CG −2.4867 11.0463 −34.3877 690 PHE127 CD1−1.8966 9.9921 −33.6854 691 PHE127 CD2 −2.7169 12.2688 −33.7507 692PHE127 CE1 −1.54 10.1607 −32.345 693 PHE127 CE2 −2.3612 12.4355 −32.4099694 PHE127 CZ −1.7752 11.3805 −31.7056 695 TYR131 N −9.2595 13.5067−37.4336 696 TYR131 CA −8.2461 13.9236 −36.4549 697 TYR131 C −8.686815.1692 −35.6672 698 TYR131 O −8.571 15.2393 −34.441 699 TYR131 CB−6.8845 14.1573 −37.1456 700 TYR131 CG −6.0123 15.0932 −36.3094 701TYR131 CD1 −5.3854 14.636 −35.1466 702 TYR131 CD2 −5.8567 16.4228−36.7094 703 TYR131 CE1 −4.671 15.5291 −34.3426 704 TYR131 CE2 −5.13817.3139 −35.9092 705 TYR131 CZ −4.5609 16.8715 −34.7166 706 TYR131 OH−3.879 17.7694 −33.9043 707 ALA132 N −9.0168 16.2679 −36.4355 708 ALA132CA −9.5328 17.5262 −35.8823 709 ALA132 C −8.5924 18.3499 −34.9607 710ALA132 O −8.709 19.5738 −34.8854 711 ALA132 CB −10.8532 17.2515 −35.1364712 GLY133 N −7.752 17.6291 −34.1455 713 GLY133 CA −6.9354 18.218−33.0956 714 GLY133 C −7.6072 18.1022 −31.7283 715 GLY133 O −7.563919.0118 −30.9002 716 ILE134 N −8.1599 16.8637 −31.4524 717 ILE134 CA−8.9173 16.6142 −30.2263 718 ILE134 C −8.8182 15.1192 −29.8913 719ILE134 O −8.8813 14.2514 −30.7603 720 ILE134 CB −10.3593 17.1524−30.3084 721 ILE134 CG1 −11.0592 17.0133 −28.9422 722 ILE134 CG2−11.1426 16.4052 −31.4039 723 ILE134 CD1 −12.4559 17.662 −28.9913 724PHE135 N −8.7247 14.8401 −28.5422 725 PHE135 CA −8.6863 13.4695 −28.0316726 PHE135 C −9.4508 13.4417 −26.7029 727 PHE135 O −9.6315 14.454−26.0215 728 PHE135 CB −7.2428 12.9517 −27.8844 729 PHE135 CG −6.584412.915 −29.2601 730 PHE135 CD1 −5.5275 13.7812 −29.5524 731 PHE135 CD2−7.0427 12.0197 −30.2306 732 PHE135 CE1 −4.95 13.7723 −30.8247 733PHE135 CE2 −6.4702 12.0164 −31.5053 734 PHE135 CZ −5.4282 12.898−31.8044 735 ARG136 N −9.8698 12.1836 −26.3098 736 ARG136 CA −10.614811.9692 −25.0723 737 ARG136 C −10.1408 10.7089 −24.3312 738 ARG136 O−9.6374 9.7405 −24.9031 739 ARG136 CB −12.1337 11.9063 −25.3183 740ARG136 CG −12.4699 10.6748 −26.1801 741 ARG136 CD −13.9424 10.2825−25.9652 742 ARG136 NE −14.3499 9.3766 −27.0232 743 ARG136 CZ −15.43769.5841 −27.7076 744 ARG136 NH1 −15.762 8.7525 −28.6524 745 ARG136 NH2−16.2059 10.6056 −27.4648 746 PHE137 N −10.3449 10.7858 −22.9627 747PHE137 CA −9.8043 9.8128 −22.011 748 PHE137 C −10.7722 9.7426 −20.8275749 PHE137 O −11.4967 10.6948 −20.5245 750 PHE137 CB −8.4306 10.2588−21.4726 751 PHE137 CG −7.3656 10.217 −22.5633 752 PHE137 CD1 −6.737111.3995 −22.9629 753 PHE137 CD2 −7.0118 9.0021 −23.1568 754 PHE137 CE1−5.7247 11.363 −23.9254 755 PHE137 CE2 −6.0012 8.966 −24.1212 756 PHE137CZ −5.3502 10.1442 −24.4974 757 TRP138 N −10.7345 8.5614 −20.1081 758TRP138 CA −11.5544 8.3889 −18.9132 759 TRP138 C −10.6682 8.1648 −17.6812760 TRP138 O −9.5891 7.5783 −17.7445 761 TRP138 CB −12.5733 7.2508−19.0873 762 TRP138 CG −13.8737 7.8635 −19.5123 763 TRP138 CD1 −14.19258.302 −20.7399 764 TRP138 CD2 −15.0672 8.1022 −18.628 765 TRP138 NE1−15.4138 8.7761 −20.7391 766 TRP138 CE2 −15.9622 8.6761 −19.5107 767TRP138 CE3 −15.3643 7.8732 −17.2856 768 TRP138 CZ2 −17.2375 9.072−19.1083 769 TRP138 CZ3 −16.6435 8.2685 −16.8761 770 TRP138 CH2 −17.55338.8666 −17.7597 771 PHE139 N −11.2525 8.5736 −16.4965 772 PHE139 CA−10.6047 8.4027 −15.1966 773 PHE139 C −11.6784 8.0116 −14.1835 774PHE139 O −12.8384 8.4183 −14.2702 775 PHE139 CB −9.764 9.6274 −14.7891776 PHE139 CG −8.9383 10.0973 −15.9836 777 PHE139 CD1 −9.3498 11.2112−16.7203 778 PHE139 CD2 −7.7763 9.4121 −16.3481 779 PHE139 CE1 −8.615111.6229 −17.8355 780 PHE139 CE2 −7.043 9.8219 −17.4648 781 PHE139 CZ−7.4627 10.9269 −18.2099 782 TRP140 N −11.2181 7.2355 −13.1403 783TRP140 CA −12.0124 7.0661 −11.921 784 TRP140 C −11.6556 8.2532 −11.0208785 TRP140 O −10.5299 8.754 −11.0265 786 TRP140 CB −11.6066 5.7386−11.2498 787 TRP140 CG −12.07 5.687 −9.8235 788 TRP140 CD1 −13.20495.1441 −9.3567 789 TRP140 CD2 −11.3253 6.2514 −8.6444 790 TRP140 NE1−13.2639 5.2892 −8.0555 791 TRP140 CE2 −12.1694 5.9322 −7.598 792 TRP140CE3 −10.1209 6.9325 −8.4733 793 TRP140 CZ2 −11.8562 6.2641 −6.2802 794TRP140 CZ3 −9.8106 7.2898 −7.1556 795 TRP140 CH2 −10.6545 6.9571 −6.0853796 HIS141 N −12.6722 8.7017 −10.2057 797 HIS141 CA −12.5073 9.8245−9.2918 798 HIS141 C −13.4191 9.5704 −8.0813 799 HIS141 O −14.62469.8188 −8.0798 800 HIS141 CB −12.934 11.1155 −10.0147 801 HIS141 CG−12.0532 11.287 −11.2152 802 HIS141 ND1 −10.8127 11.7037 −11.1111 803HIS141 CD2 −12.4213 11.027 −12.4836 804 HIS141 CE1 −10.3183 11.7319−12.3074 805 HIS141 NE2 −11.1851 11.36 −13.1359 806 TYR142 N −12.7958.9359 −7.0187 807 TYR142 CA −13.3433 9.0058 −5.6555 808 TYR142 C−14.7314 8.3527 −5.5148 809 TYR142 O −15.4898 8.5952 −4.5781 810 TYR142CB −13.397 10.4616 −5.1484 811 TYR142 CG −12.0073 11.0876 −5.0786 812TYR142 CD1 −11.2482 11.2589 −6.2396 813 TYR142 CD2 −11.4964 11.4996−3.8452 814 TYR142 CE1 −9.9943 11.8714 −6.1724 815 TYR142 CE2 −10.247312.1222 −3.7792 816 TYR142 CZ −9.5014 12.319 −4.9439 817 TYR142 OH−8.2703 12.9606 −4.8804 818 GLY143 N −14.9795 7.3789 −6.4539 819 GLY143CA −16.2053 6.619 −6.5102 820 GLY143 C −16.7664 6.5699 −7.9246 821GLY143 O −17.2517 5.5321 −8.3758 822 ASN144 N −16.8015 7.7865 −8.5793823 ASN144 CA −17.3743 7.914 −9.9166 824 ASN144 C −16.2826 7.8175−10.9978 825 ASN144 O −15.0873 7.9838 −10.7567 826 ASN144 CB −18.10959.2584 −10.0685 827 ASN144 CG −19.2871 9.3093 −9.1396 828 ASN144 OD1−19.3421 10.171 −8.2779 829 ASN144 ND2 −20.2454 8.3803 −9.3069 830TRP145 N −16.775 7.5382 −12.2662 831 TRP145 CA −15.9231 7.6557 −13.4515832 TRP145 C −16.1906 9.0373 −14.058 833 TRP145 O −17.311 9.5466−14.0413 834 TRP145 CB −15.9475 6.4578 −14.4157 835 TRP145 CG −14.75515.6159 −14.0635 836 TRP145 CD1 −14.6017 4.8703 −12.9577 837 TRP145 CD2−13.5001 5.4609 −14.88 838 TRP145 NE1 −13.4297 4.2851 −12.9715 839TRP145 CE2 −12.7449 4.6195 −14.0844 840 TRP145 CE3 −13.0513 5.9493−16.1065 841 TRP145 CZ2 −11.4595 4.2199 −14.4501 842 TRP145 CZ3 −11.76775.5395 −16.4885 843 TRP145 CH2 −10.9842 4.7093 −15.6732 844 VAL146 N−15.0943 9.6111 −14.6657 845 VAL146 CA −15.1323 10.9329 −15.288 846VAL146 C −14.4039 10.7866 −16.6377 847 VAL146 O −13.3481 10.1608−16.7543 848 VAL146 CB −14.5062 12.0252 −14.4016 849 VAL146 CG1 −14.562713.3836 −15.1259 850 VAL146 CG2 −15.2933 12.1222 −13.0812 851 PRO147 N−15.0407 11.4243 −17.688 852 PRO147 CA −14.4421 11.5445 −19.0197 853PRO147 C −13.7611 12.9212 −19.0753 854 PRO147 O −14.2574 13.9182−18.5488 855 PRO147 CB −15.7135 11.6846 −19.8787 856 PRO147 CG −16.829612.2116 −18.9477 857 PRO147 CD −16.3539 12.0027 −17.496 858 VAL148 N−12.5849 12.9549 −19.7972 859 VAL148 CA −11.8514 14.1932 −20.0352 860VAL148 C −11.4656 14.2299 −21.5245 861 VAL148 O −11.0249 13.2515−22.1259 862 VAL148 CB −10.5713 14.2143 −19.1768 863 VAL148 CG1 −9.897715.597 −19.2662 864 VAL148 CG2 −10.922 13.9061 −17.7098 865 VAL149 N−11.6336 15.4826 −22.0884 866 VAL149 CA −11.3107 15.8132 −23.4772 867VAL149 C −10.0416 16.689 −23.4257 868 VAL149 O −9.863 17.5286 −22.5332869 VAL149 CB −12.4666 16.541 −24.1919 870 VAL149 CG1 −12.0679 16.8561−25.6467 871 VAL149 CG2 −13.7176 15.6425 −24.1975 872 ILE150 N −9.158416.5193 −24.4747 873 ILE150 CA −7.8397 17.1654 −24.4977 874 ILE150 C−7.3708 17.3781 −25.9591 875 ILE150 O −7.7728 16.6718 −26.8855 876ILE150 CB −6.8268 16.3699 −23.645 877 ILE150 CG1 −6.7359 14.8871−24.0676 878 ILE150 CG2 −5.4384 17.0398 −23.6387 879 ILE150 CD1 −6.036914.7232 −25.4318 880 ASP151 N −6.4562 18.4073 −26.1257 881 ASP151 CA−5.7667 18.6717 −27.3943 882 ASP151 C −4.3473 18.0589 −27.3541 883ASP151 O −3.8018 17.6815 −26.3105 884 ASP151 CB −5.6562 20.196 −27.5904885 ASP151 CG −4.8288 20.8034 −26.4927 886 ASP151 OD1 −3.5843 20.8953−26.6713 887 ASP151 OD2 −5.4241 21.2042 −25.4574 888 ASP152 N −3.691318.0528 −28.5621 889 ASP152 CA −2.4247 17.3595 −28.815 890 ASP152 C−1.2286 18.3059 −28.9933 891 ASP152 O −0.1476 17.9427 −29.4503 892ASP152 CB −2.5075 16.1388 −29.7539 893 ASP152 CG −3.1537 16.4648−31.0697 894 ASP152 OD1 −2.4711 16.2908 −32.1139 895 ASP152 OD2 −4.341316.8882 −31.068 896 ARG153 N −1.4181 19.5454 −28.4233 897 ARG153 CA−0.3124 20.46 −28.1713 898 ARG153 C −0.269 20.0286 −26.8111 899 ARG153 O−0.4419 19.9335 −25.8052 900 ARG153 CB −0.8267 21.9081 −28.0912 901ARG153 CG −1.4103 22.3167 −29.457 902 ARG153 CD −2.9178 22.6007 −29.3184903 ARG153 NE −3.1155 23.7076 −28.3995 904 ARG153 CZ −4.2574 23.8944−27.8041 905 ARG153 NH1 −5.2654 23.1038 −28.0233 906 ARG153 NH2 −4.395924.8896 −26.9796 907 LEU154 N 1.6045 19.6786 −26.8477 908 LEU154 CA2.3415 19.1173 −25.7092 909 LEU154 C 3.5418 20.0371 −25.3873 910 LEU154O 3.993 20.806 −26.2439 911 LEU154 CB 2.8149 17.6876 −26.038 912 LEU154CG 1.6904 16.8972 −26.7388 913 LEU154 CD1 2.2113 15.5079 −27.1503 914LEU154 CD2 0.4791 16.7385 −25.7994 915 PRO155 N 4.1317 19.919 −24.1374916 PRO155 CA 5.2572 20.7795 −23.7535 917 PRO155 C 6.5862 20.1545−24.2212 918 PRO155 O 7.092 19.1556 −23.7017 919 PRO155 CB 5.171320.6814 −22.217 920 PRO155 CG 4.3153 19.4413 −21.8727 921 PRO155 CD3.5972 18.9944 −23.1615 922 LEU162 N 9.1036 17.1652 −22.7622 923 LEU162CA 9.5653 17.3634 −21.3825 924 LEU162 C 8.9453 16.3509 −20.3944 925LEU162 O 9.3738 16.2255 −19.2479 926 LEU162 CB 9.1718 18.7986 −20.9763927 LEU162 CG 9.5659 19.7972 −22.085 928 LEU162 CD1 8.9469 21.1771−21.7988 929 LEU162 CD2 11.0986 19.9205 −22.171 930 VAL163 N 7.805915.7359 −20.8548 931 VAL163 CA 6.9001 14.9282 −20.0167 932 VAL163 C6.8592 13.4935 −20.5742 933 VAL163 O 7.6291 13.1371 −21.4753 934 VAL163CB 5.5354 15.6534 −20.0225 935 VAL163 CG1 4.4405 14.9111 −20.8157 936VAL163 CG2 5.0723 15.9333 −18.5813 937 PHE164 N 5.9919 12.5981 −19.9705938 PHE164 CA 6.0594 11.1646 −20.2933 939 PHE164 C 5.6914 10.9875−21.7803 940 PHE164 O 4.8581 11.6769 −22.3624 941 PHE164 CB 5.03310.3666 −19.4641 942 PHE164 CG 5.4169 10.3322 −17.9883 943 PHE164 CD14.5025 10.7562 −17.0208 944 PHE164 CD2 6.6779 9.8748 −17.5988 945 PHE164CE1 4.8585 10.7499 −15.6698 946 PHE164 CE2 7.0391 9.8751 −16.2491 947PHE164 CZ 6.1318 10.3218 −15.2852 948 VAL165 N 6.3903 9.9741 −22.4123949 VAL165 CA 6.3353 9.7874 −23.8582 950 VAL165 C 6.8056 8.3647 −24.1952951 VAL165 O 7.4217 7.6684 −23.3901 952 VAL165 CB 7.2052 10.8184−24.6081 953 VAL165 CG1 6.5458 12.2098 −24.5778 954 VAL165 CG2 8.611910.8981 −23.9844 955 SER166 N 6.4785 7.9386 −25.4751 956 SER166 CA7.1328 6.7639 −26.0454 957 SER166 C 6.838 6.5724 −27.5437 958 SER166 O5.686 6.5221 −27.96 959 SER166 CB 6.7683 5.4809 −25.2727 960 SER166 OG7.7074 4.4486 −25.5853 961 PHE173 N 2.2461 8.4031 −28.877 962 PHE173 CA1.3314 9.4965 −28.6476 963 PHE173 C 0.336 9.3901 −27.514 964 PHE173 O−0.4975 10.2913 −27.372 965 PHE173 CB 0.6858 10.0229 −29.9456 966 PHE173CG 1.7267 10.7609 −30.7817 967 PHE173 CD1 1.8293 10.5041 −32.1511 968PHE173 CD2 2.5797 11.6915 −30.1812 969 PHE173 CE1 2.8202 11.1379−32.9053 970 PHE173 CE2 3.5614 12.3352 −30.9387 971 PHE173 CZ 3.686512.0519 −32.3009 972 TRP174 N −0.2464 8.1445 −27.29 973 TRP174 CA−1.3832 8.0441 −26.3625 974 TRP174 C −0.9695 8.3698 −24.9116 975 TRP174O −1.7629 8.8377 −24.0928 976 TRP174 CB −2.0615 6.6656 −26.4833 977TRP174 CG −1.251 5.612 −25.7898 978 TRP174 CD1 −0.0053 5.2237 −26.1002979 TRP174 CD2 −1.6945 4.7987 −24.6045 980 TRP174 NE1 0.3806 4.2861−25.2719 981 TRP174 CE2 −0.5919 3.9966 −24.3848 982 TRP174 CE3 −2.8474.736 −23.8226 983 TRP174 CZ2 −0.5679 3.042 −23.3686 984 TRP174 CZ3−2.8211 3.7981 −22.7832 985 TRP174 CH2 −1.7146 2.9625 −22.5695 986GLY175 N 0.319 8.0074 −24.5881 987 GLY175 CA 0.8548 8.0482 −23.229 988GLY175 C 0.924 9.5013 −22.7453 989 GLY175 O 0.4744 9.8591 −21.6563 990ALA176 N 1.5527 10.3575 −23.6355 991 ALA176 CA 1.8162 11.7463 −23.2667992 ALA176 C 0.4778 12.4832 −23.1036 993 ALA176 O 0.3087 13.3923−22.2917 994 ALA176 CB 2.6081 12.4016 −24.4134 995 LEU177 N −0.486212.0977 −24.0175 996 LEU177 CA −1.8072 12.7043 −24.039 997 LEU177 C−2.6026 12.2801 −22.7889 998 LEU177 O −3.3645 13.0632 −22.2149 999LEU177 CB −2.5591 12.1878 −25.2811 1000 LEU177 CG −2.1824 13.0175−26.5242 1001 LEU177 CD1 −2.8503 12.4028 −27.7689 1002 LEU177 CD2−2.6658 14.4711 −26.3557 1003 LEU178 N −2.4445 10.9664 −22.3884 1004LEU178 CA −3.1132 10.4544 −21.1906 1005 LEU178 C −2.5251 11.1643−19.9513 1006 LEU178 O −3.234 11.5138 −19.0034 1007 LEU178 CB −2.91778.9296 −21.0891 1008 LEU178 CG −3.8563 8.3476 −20.0123 1009 LEU178 CD1−5.3264 8.6533 −20.3596 1010 LEU178 CD2 −3.6637 6.822 −19.9332 1011GLU179 N −1.1524 11.3234 −19.9572 1012 GLU179 CA −0.4475 11.9849−18.8542 1013 GLU179 C −0.9306 13.4538 −18.7813 1014 GLU179 O −1.21713.995 −17.7092 1015 GLU179 CB 1.0603 11.9436 −19.1576 1016 GLU179 CG1.836 12.3442 −17.8903 1017 GLU179 CD 2.7056 13.5265 −18.1976 1018GLU179 OE1 3.9506 13.3429 −18.2297 1019 GLU179 OE2 2.1484 14.6392−18.399 1020 LYS180 N −1.0611 14.1019 −20.0001 1021 LYS180 CA −1.529415.4849 −20.058 1022 LYS180 C −2.9863 15.5584 −19.5596 1023 LYS180 O−3.4062 16.527 −18.9195 1024 LYS180 CB −1.4866 15.9011 −21.5416 1025LYS180 CG −1.9217 17.3697 −21.7058 1026 LYS180 CD −1.7157 17.8037−23.1693 1027 LYS180 CE −2.086 19.2905 −23.326 1028 LYS180 NZ −3.451919.4078 −23.8566 1029 ALA181 N −3.8236 14.534 −19.9589 1030 ALA181 CA−5.227 14.5397 −19.5633 1031 ALA181 C −5.3504 14.3395 −18.0377 1032ALA181 O −6.2726 14.8376 −17.3882 1033 ALA181 CB −5.9142 13.3553−20.2685 1034 TYR182 N −4.3968 13.5147 −17.4674 1035 TYR182 CA −4.337813.3084 −16.014 1036 TYR182 C −3.911 14.6397 −15.3604 1037 TYR182 O−4.4286 15.0613 −14.3233 1038 TYR182 CB −3.2428 12.2534 −15.7604 1039TYR182 CG −3.7672 11.0667 −14.9534 1040 TYR182 CD1 −2.8826 10.3532−14.1401 1041 TYR182 CD2 −5.1102 10.6836 −15.0169 1042 TYR182 CE1−3.3303 9.249 −13.4101 1043 TYR182 CE2 −5.5616 9.5844 −14.2811 1044TYR182 CZ −4.6681 8.8552 −13.4923 1045 TYR182 OH −5.1083 7.7388 −12.7921046 ALA183 N −2.9084 15.3364 −16.0158 1047 ALA183 CA −2.4983 16.6457−15.5238 1048 ALA183 C −3.6901 17.6177 −15.5866 1049 ALA183 O −3.864318.4841 −14.7264 1050 ALA183 CB −1.384 17.1672 −16.4469 1051 LYS184 N−4.5087 17.4934 −16.6977 1052 LYS184 CA −5.611 18.422 −16.9143 1053LYS184 C −6.6679 18.2596 −15.8071 1054 LYS184 O −7.2776 19.2443 −15.3831055 LYS184 CB −6.2294 18.0837 −18.2832 1056 LYS184 CG −7.201 19.2001−18.7066 1057 LYS184 CD −7.6414 18.9677 −20.1633 1058 LYS184 CE −8.736119.9852 −20.5324 1059 LYS184 NZ −10.0419 19.4944 −20.0694 1060 LEU185 N−6.9762 16.9651 −15.4203 1061 LEU185 CA −7.9755 16.7511 −14.3701 1062LEU185 C −7.4202 17.1729 −12.997 1063 LEU185 O −8.1656 17.635 −12.13121064 LEU185 CB −8.3535 15.2591 −14.3817 1065 LEU185 CG −9.887 15.1102−14.4244 1066 LEU185 CD1 −10.4851 15.9782 −15.5488 1067 LEU185 CD2−10.2408 13.6361 −14.6884 1068 SER186 N −6.0696 16.9426 −12.7748 1069SER186 CA −5.4961 17.2909 −11.4717 1070 SER186 C −5.3992 18.8264−11.3662 1071 SER186 O −5.5294 19.4181 −10.296 1072 SER186 CB −4.091816.6661 −11.3442 1073 SER186 OG −3.2145 17.1663 −12.3577 1074 GLY187 N−5.0223 19.4634 −12.5324 1075 GLY187 CA −5.2652 20.8786 −12.7556 1076GLY187 C −4.0857 21.5354 −13.4597 1077 GLY187 O −4.2258 22.4645 −14.25111078 SER188 N −2.8658 21.077 −13.0129 1079 SER188 CA −1.5882 21.562−13.5121 1080 SER188 C −0.6525 20.3481 −13.5519 1081 SER188 O −0.769719.4057 −12.7666 1082 SER188 CB −0.9967 22.6529 −12.6005 1083 SER188 OG−1.8848 23.7722 −12.5576 1084 TYR189 N 0.4309 20.4751 −14.4103 1085TYR189 CA 1.394 19.3635 −14.5345 1086 TYR189 C 2.1028 19.1407 −13.18751087 TYR189 O 2.5564 18.0528 −12.8423 1088 TYR189 CB 2.4205 19.6809−15.6392 1089 TYR189 CG 1.7359 19.639 −17.0026 1090 TYR189 CD1 1.696620.7877 −17.7958 1091 TYR189 CD2 1.1439 18.4599 −17.4631 1092 TYR189 CE10.9697 20.7938 −18.9882 1093 TYR189 CE2 0.4434 18.4535 −18.6726 1094TYR189 CZ 0.3372 19.627 −19.4238 1095 TYR189 OH −0.3958 19.6344 −20.60381096 GLU190 N 2.2353 20.2856 −12.4271 1097 GLU190 CA 2.9165 20.2945−11.1371 1098 GLU190 C 2.0888 19.4928 −10.1168 1099 GLU190 O 2.611718.9198 −9.1614 1100 GLU190 CB 2.9576 21.7516 −10.642 1101 GLU190 CG3.8849 21.8584 −9.417 1102 GLU190 CD 5.0422 22.7653 −9.7201 1103 GLU190OE1 5.8825 22.393 −10.5826 1104 GLU190 OE2 5.1173 23.8523 −9.0882 1105ASP191 N 0.7198 19.4753 −10.3209 1106 ASP191 CA −0.1724 18.7469 −9.42371107 ASP191 C −0.2456 17.2321 −9.7198 1108 ASP191 O −1.1273 16.5148−9.243 1109 ASP191 CB −1.5988 19.335 −9.455 1110 ASP191 CG −1.604320.8365 −9.5147 1111 ASP191 OD1 −2.4983 21.3884 −10.2095 1112 ASP191 OD2−0.7159 21.4698 −8.8827 1113 LEU192 N 0.819 16.7221 −10.4365 1114 LEU192CA 1.1802 15.3111 −10.4241 1115 LEU192 C 2.3645 15.0764 −9.4578 1116LEU192 O 2.6051 13.967 −8.9756 1117 LEU192 CB 1.606 14.9339 −11.85841118 LEU192 CG 0.6433 15.546 −12.899 1119 LEU192 CD1 1.1591 15.2637−14.3222 1120 LEU192 CD2 −0.7731 14.9614 −12.7367 1121 GLN193 N 3.222516.1521 −9.2984 1122 GLN193 CA 4.4874 16.0024 −8.5836 1123 GLN193 C4.1847 15.9215 −7.0888 1124 GLN193 O 3.7016 16.8552 −6.4531 1125 GLN193CB 5.4245 17.2036 −8.8285 1126 GLN193 CG 5.1253 17.9062 −10.168 1127GLN193 CD 6.0074 19.112 −10.3215 1128 GLN193 OE1 6.5849 19.3114 −11.37721129 GLN193 NE2 6.126 19.9311 −9.2612 1130 SER194 N 4.4584 14.6824−6.5404 1131 SER194 CA 4.1404 14.3758 −5.1598 1132 SER194 C 2.86613.5587 −4.9666 1133 SER194 O 2.5222 13.2064 −3.8381 1134 SER194 CB4.3567 15.493 −4.1172 1135 SER194 OG 3.1377 16.2063 −3.8864 1136 GLY195N 2.176 13.2208 −6.1141 1137 GLY195 CA 1.1358 12.206 −6.0658 1138 GLY195C 1.7882 10.822 −6.0921 1139 GLY195 O 2.8873 10.6093 −6.6065 1140 GLU199N −2.5121 6.1465 −5.8806 1141 GLU199 CA −3.5773 7.0891 −6.2252 1142GLU199 C −3.8566 6.9376 −7.7303 1143 GLU199 O −4.9993 6.8618 −8.18121144 GLU199 CB −3.046 8.5117 −5.9794 1145 GLU199 CG −2.8762 8.7261−4.4654 1146 GLU199 CD −1.8341 9.7648 −4.1654 1147 GLU199 OE1 −1.6310.6852 −5.0024 1148 GLU199 OE2 −1.2058 9.6511 −3.0809 1149 ALA200 N−2.7213 6.902 −8.5278 1150 ALA200 CA −2.842 6.7555 −9.9605 1151 ALA200 C−3.45 5.3988 −10.3097 1152 ALA200 O −4.3298 5.2754 −11.1633 1153 ALA200CB −1.4357 6.8497 −10.5805 1154 LEU201 N −2.922 4.3129 −9.635 1155LEU201 CA −3.3751 2.957 −9.9427 1156 LEU201 C −4.9075 2.8846 −9.75471157 LEU201 O −5.6129 2.2233 −10.5246 1158 LEU201 CB −2.7345 2.0008−8.9179 1159 LEU201 CG −2.2696 0.7033 −9.6083 1160 LEU201 CD1 −1.7303−0.2762 −8.5491 1161 LEU201 CD2 −3.4383 0.0413 −10.3625 1162 VAL202 N−5.4127 3.5437 −8.6455 1163 VAL202 CA −6.8497 3.6253 −8.4177 1164 VAL202C −7.5073 4.5069 −9.4862 1165 VAL202 O −8.4947 4.0924 −10.1074 1166VAL202 CB −7.1239 4.2445 −7.0323 1167 VAL202 CG1 −8.6414 4.274 −6.76491168 VAL202 CG2 −6.4384 3.4126 −5.9324 1169 ASP203 N −6.9576 5.7508−9.7191 1170 ASP203 CA −7.662 6.72 −10.568 1171 ASP203 C −7.7643 6.2123−12.0267 1172 ASP203 O −8.6495 6.6136 −12.7897 1173 ASP203 CB −6.93128.0729 −10.5074 1174 ASP203 CG −7.5182 8.8924 −9.3946 1175 ASP203 OD1−7.1833 8.6159 −8.2117 1176 ASP203 OD2 −8.3092 9.8239 −9.7007 1177PHE204 N −6.7745 5.3288 −12.4181 1178 PHE204 CA −6.7787 4.6718 −13.72091179 PHE204 C −7.5039 3.3039 −13.7173 1180 PHE204 O −7.4301 2.5569−14.6962 1181 PHE204 CB −5.3233 4.4568 −14.1833 1182 PHE204 CG −4.79065.6489 −14.9755 1183 PHE204 CD1 −5.63 6.3842 −15.8175 1184 PHE204 CD2−3.4431 6.0012 −14.8599 1185 PHE204 CE1 −5.1116 7.4353 −16.5787 1186PHE204 CE2 −2.9247 7.0537 −15.619 1187 PHE204 CZ −3.7573 7.7662 −16.48581188 THR205 N −8.3016 2.9986 −12.63 1189 THR205 CA −8.952 1.6829−12.5554 1190 THR205 C −10.2705 1.7051 −11.7627 1191 THR205 O −11.2951.18 −12.1962 1192 THR205 CB −7.9841 0.6757 −11.8991 1193 THR205 OG1−6.7587 0.6175 −12.6342 1194 THR205 CG2 −8.6165 −0.7291 −11.8676 1195GLY206 N −10.175 2.1806 −10.4697 1196 GLY206 CA −11.3324 2.192 −9.58851197 GLY206 C −11.5494 0.908 −8.7917 1198 GLY206 O −12.5236 0.7578−8.0566 1199 GLY207 N −10.5234 −0.0067 −8.9016 1200 GLY207 CA −10.3755−1.0644 −7.9225 1201 GLY207 C −9.6759 −0.488 −6.6908 1202 GLY207 O−9.3188 0.6865 −6.6208 1203 VAL208 N −9.4719 −1.4096 −5.6779 1204 VAL208CA −8.7137 −1.0176 −4.4842 1205 VAL208 C −7.2252 −1.2657 −4.7591 1206VAL208 O −6.8341 −2.3528 −5.1916 1207 VAL208 CB −9.1628 −1.8 −3.23431208 VAL208 CG1 −8.4598 −1.2244 −1.9901 1209 VAL208 CG2 −10.6865 −1.6664−3.0526 1210 ASN220 N 0.5391 −14.5311 5.7482 1211 ASN220 CA −0.345−15.0597 4.7114 1212 ASN220 C −0.1301 −14.4188 3.3248 1213 ASN220 O−0.8992 −14.5951 2.3761 1214 ASN220 CB −1.8072 −14.8271 5.1425 1215ASN220 CG −2.0916 −13.3553 5.2608 1216 ASN220 OD1 −2.825 −12.8137 4.45121217 ASN220 ND2 −1.5115 −12.6891 6.2753 1218 LEU221 N 1.0815 −13.77133.1652 1219 LEU221 CA 1.2715 −12.8164 2.0571 1220 LEU221 C 1.2799−13.5607 0.7046 1221 LEU221 O 0.9346 −13.036 −0.3535 1222 LEU221 CB2.6218 −12.1004 2.259 1223 LEU221 CG 2.9041 −11.0458 1.1678 1224 LEU221CD1 3.4846 −11.7071 −0.0966 1225 LEU221 CD2 1.6457 −10.2237 0.8287 1226TRP222 N 1.7467 −14.8606 0.7729 1227 TRP222 CA 1.8026 −15.7229 −0.40881228 TRP222 C 0.3893 −16.1528 −0.8382 1229 TRP222 O 0.1312 −16.4911−1.9975 1230 TRP222 CB 2.6551 −16.9633 −0.0895 1231 TRP222 CG 3.514−17.3207 −1.269 1232 TRP222 CD1 3.9296 −18.551 −1.6056 1233 TRP222 CD24.0798 −16.3703 −2.2906 1234 TRP222 NE1 4.6819 −18.4953 −2.6768 1235TRP222 CE2 4.8057 −17.2219 −3.1023 1236 TRP222 CE3 4.0008 −14.9958−2.5113 1237 TRP222 CZ2 5.5389 −16.7516 −4.1911 1238 TRP222 CZ3 4.7089−14.5196 −3.6215 1239 TRP222 CH2 5.4654 −15.3749 −4.4357 1240 ASP223 N−0.5353 −16.2792 0.1887 1241 ASP223 CA −1.9239 −16.5685 −0.1432 1242ASP223 C −2.5885 −15.3166 −0.7289 1243 ASP223 O −3.4409 −15.4233 −1.61131244 ASP223 CB −2.6696 −16.9075 1.1623 1245 ASP223 CG −1.9071 −17.92061.9661 1246 ASP223 OD1 −2.0616 −19.1383 1.6831 1247 ASP223 OD2 −1.1616−17.4999 2.8907 1248 ILE224 N −2.193 −14.0971 −0.2059 1249 ILE224 CA−2.7525 −12.8492 −0.744 1250 ILE224 C −2.3698 −12.7895 −2.2384 1251ILE224 O −3.2148 −12.5541 −3.1062 1252 ILE224 CB −1.9614 −11.7006 −0.081253 ILE224 CG1 −2.0125 −11.7554 1.4607 1254 ILE224 CG2 −2.415 −10.3245−0.6073 1255 ILE224 CD1 −3.3687 −11.2443 1.9805 1256 LEU225 N −1.0318−13.0064 −2.5371 1257 LEU225 CA −0.5436 −12.7541 −3.9012 1258 LEU225 C−1.2766 −13.7007 −4.8795 1259 LEU225 O −1.6357 −13.3264 −5.9993 1260LEU225 CB 0.9715 −13.0185 −3.9213 1261 LEU225 CG 1.7156 −11.6713 −3.85611262 LEU225 CD1 1.4854 −10.9979 −2.4897 1263 LEU225 CD2 3.2213 −11.907−4.0649 1264 ILE226 N −1.4518 −15.0023 −4.4456 1265 ILE226 CA −2.0745−15.9965 −5.3171 1266 ILE226 C −3.5714 −15.6726 −5.5167 1267 ILE226 O−4.1698 −15.9481 −6.5587 1268 ILE226 CB −1.8899 −17.3793 −4.662 1269ILE226 CG1 −0.3862 −17.718 −4.6364 1270 ILE226 CG2 −2.642 −18.4549−5.4702 1271 ILE226 CD1 −0.1398 −18.9911 −3.8053 1272 GLU227 N −4.2228−15.1961 −4.388 1273 GLU227 CA −5.6282 −14.8244 −4.443 1274 GLU227 C−5.8203 −13.5496 −5.2825 1275 GLU227 O −6.8718 −13.3538 −5.8944 1276GLU227 CB −6.0543 −14.5149 −2.9947 1277 GLU227 CG −6.7645 −15.73 −2.36591278 GLU227 CD −5.8178 −16.8723 −2.1316 1279 GLU227 OE1 −5.5349 −17.1663−0.9395 1280 GLU227 OE2 −5.3604 −17.486 −3.1329 1281 ALA228 N −4.7777−12.6483 −5.2521 1282 ALA228 CA −4.776 −11.455 −6.0904 1283 ALA228 C−4.5622 −11.8767 −7.5598 1284 ALA228 O −5.1727 −11.3474 −8.4919 1285ALA228 CB −3.5912 −10.5744 −5.6525 1286 THR229 N −3.6251 −12.867 −7.79221287 THR229 CA −3.3162 −13.2583 −9.1709 1288 THR229 C −4.5689 −13.8572−9.8218 1289 THR229 O −4.9097 −13.5481 −10.9646 1290 THR229 CB −2.119−14.2272 −9.2376 1291 THR229 OG1 −2.3184 −15.3492 −8.3756 1292 THR229CG2 −0.825 −13.4907 −8.8429 1293 TYR230 N −5.2918 −14.763 −9.0662 1294TYR230 CA −6.4583 −15.4248 −9.6547 1295 TYR230 C −7.7072 −14.5029−9.7559 1296 TYR230 O −8.7572 −14.8875 −10.2748 1297 TYR230 CB −6.8168−16.6984 −8.8549 1298 TYR230 CG −7.4666 −16.426 −7.4941 1299 TYR230 CD1−6.9898 −17.0901 −6.3609 1300 TYR230 CD2 −8.5379 −15.5367 −7.3628 1301TYR230 CE1 −7.6126 −16.9041 −5.1237 1302 TYR230 CE2 −9.1241 −15.2992−6.118 1303 TYR230 CZ −8.6664 −15.9953 −4.9973 1304 TYR230 OH −9.2605−15.7837 −3.7595 1305 ASN231 N −7.5398 −13.2328 −9.2411 1306 ASN231 CA−8.4707 −12.1217 −9.4628 1307 ASN231 C −8.1441 −11.3886 −10.7844 1308ASN231 O −8.9255 −10.5879 −11.3015 1309 ASN231 CB −8.3969 −11.1441−8.2751 1310 ASN231 CG −9.4706 −11.4856 −7.2813 1311 ASN231 OD1 −9.169−11.7934 −6.1402 1312 ASN231 ND2 −10.7455 −11.4325 −7.7086 1313 ARG232 N−6.8621 −11.5676 −11.2662 1314 ARG232 CA −6.3853 −10.8097 −12.4064 1315ARG232 C −6.0159 −9.4036 −11.9561 1316 ARG232 O −6.1526 −8.4293 −12.69581317 ARG232 CB −7.3543 −10.8028 −13.6066 1318 ARG232 CG −7.3407 −12.1783−14.2961 1319 ARG232 CD −8.3543 −12.1759 −15.4551 1320 ARG232 NE −8.1377−13.349 −16.2813 1321 ARG232 CZ −7.7623 −13.2361 −17.5225 1322 ARG232NH1 −7.5672 −14.3083 −18.2308 1323 ARG232 NH2 −7.5763 −12.0707 −18.06911324 THR233 N −5.4344 −9.3387 −10.7042 1325 THR233 CA −5.0346 −8.0619−10.1287 1326 THR233 C −3.7566 −7.5873 −10.8289 1327 THR233 O −2.8805−8.3575 −11.2235 1328 THR233 CB −4.8108 −8.1256 −8.6048 1329 THR233 OG1−3.7279 −9.0059 −8.2917 1330 THR233 CG2 −6.0987 −8.5817 −7.8934 1331LEU234 N −3.6383 −6.2111 −10.9129 1332 LEU234 CA −2.4212 −5.6082−11.4445 1333 LEU234 C −1.4991 −5.4025 −10.2343 1334 LEU234 O −1.7828−4.5966 −9.3461 1335 LEU234 CB −2.7335 −4.2456 −12.0927 1336 LEU234 CG−3.805 −4.3977 −13.1899 1337 LEU234 CD1 −4.1814 −3.0033 −13.7237 1338LEU234 CD2 −3.261 −5.2597 −14.3451 1339 ILE235 N −0.4064 −6.2504−10.1931 1340 ILE235 CA 0.6017 −6.192 −9.1379 1341 ILE235 C 1.7908−5.3356 −9.6145 1342 ILE235 O 2.2008 −5.3498 −10.779 1343 ILE235 CB1.1155 −7.6203 −8.8608 1344 ILE235 CG1 −0.0599 −8.5572 −8.5186 1345ILE235 CG2 2.1354 −7.6114 −7.7054 1346 ILE235 CD1 0.4306 −10.0168−8.4676 1347 GLY236 N 2.42 −4.6484 −8.592 1348 GLY236 CA 3.6097 −3.8503−8.8163 1349 GLY236 C 4.4661 −3.8751 −7.556 1350 GLY236 O 4.0494 −3.4215−6.4864 1351 CYS237 N 5.5388 −4.1763 −7.1446 1352 CYS237 CA 6.8369−4.8386 −7.1951 1353 CYS237 C 7.4475 −4.8061 −5.8055 1354 CYS237 O 7.484−3.758 −5.1653 1355 CYS237 CB 6.5137 −6.2914 −7.605 1356 CYS237 SG7.8892 −6.9754 −8.5747 1357 VAL250 N 18.7837 −1.06 −3.8757 1358 VAL250CA 18.7728 −1.6614 −5.1968 1359 VAL250 C 18.1563 −0.7936 −6.2755 1360VAL250 O 17.5799 0.2563 −6.0141 1361 VAL250 CB 20.2071 −2.0544 −5.61351362 VAL250 CG1 21.0983 −0.8042 −5.7459 1363 VAL250 CG2 20.1899 −2.8323−6.9436 1364 GLU251 N 18.3669 −0.2148 −7.3233 1365 GLU251 CA 17.76650.993 −7.8951 1366 GLU251 C 16.217 0.9991 −7.9433 1367 GLU251 O 15.5217−0.0022 −8.13 1368 GLU251 CB 18.3563 1.2753 −9.292 1369 GLU251 CG17.7693 0.3208 −10.3534 1370 GLU251 CD 18.039 −1.1153 −10.0041 1371GLU251 OE1 17.0546 −1.8468 −9.7159 1372 GLU251 OE2 19.2333 −1.5165−10.0181 1373 GLY252 N 15.6734 2.2759 −7.9118 1374 GLY252 CA 14.25062.4954 −7.6952 1375 GLY252 C 13.3542 2.3126 −8.9232 1376 GLY252 O12.4592 3.1102 −9.1965 1377 HIS253 N 13.5372 1.1273 −9.6114 1378 HIS253CA 12.566 0.6781 −10.6178 1379 HIS253 C 11.4865 −0.1559 −9.8957 1380HIS253 O 11.7404 −0.8619 −8.9163 1381 HIS253 CB 13.2764 −0.2088 −11.65941382 HIS253 CG 12.3129 −0.6933 −12.7051 1383 HIS253 ND1 11.7376 0.1248−13.5563 1384 HIS253 CD2 11.9466 −1.9771 −12.8817 1385 HIS253 CE110.9773 −0.5897 −14.3244 1386 HIS253 NE2 11.0523 −1.7983 −13.9931 1387ALA254 N 10.2237 −0.0612 −10.4641 1388 ALA254 CA 9.1262 −0.9262 −10.04651389 ALA254 C 8.9691 −2.0231 −11.1058 1390 ALA254 O 8.8789 −1.772−12.3048 1391 ALA254 CB 7.8177 −0.1351 −9.8715 1392 TYR255 N 8.9631−3.2945 −10.5799 1393 TYR255 CA 8.7409 −4.4746 −11.4081 1394 TYR255 C7.2757 −4.8968 −11.1662 1395 TYR255 O 6.6574 −4.578 −10.1462 1396 TYR255CB 9.7104 −5.6038 −11.0117 1397 TYR255 CG 11.1349 −5.06 −11.0372 1398TYR255 CD1 11.6245 −4.3411 −9.9433 1399 TYR255 CD2 11.9459 −5.2746−12.1545 1400 TYR255 CE1 12.9031 −3.7807 −9.9923 1401 TYR255 CE2 13.2324−4.731 −12.1946 1402 TYR255 CZ 13.7027 −3.9676 −11.1225 1403 TYR255 OH14.9665 −3.3926 −11.1806 1404 THR256 N 6.7032 −5.6869 −12.1463 1405THR256 CA 5.4017 −6.3105 −11.8927 1406 THR256 C 5.62 −7.7682 −11.46661407 THR256 O 6.704 −8.3411 −11.5791 1408 THR256 CB 4.5349 −6.2455−13.1674 1409 THR256 OG1 3.2634 −6.8578 −12.933 1410 THR256 CG2 5.2393−6.9521 −14.3426 1411 LEU257 N 4.4903 −8.3735 −10.956 1412 LEU257 CA4.4405 −9.7992 −10.6241 1413 LEU257 C 3.3352 −10.3492 −11.5298 1414LEU257 O 2.3071 −9.7058 −11.7431 1415 LEU257 CB 4.0719 −9.97 −9.13841416 LEU257 CG 4.449 −11.3805 −8.6471 1417 LEU257 CD1 4.42 −11.398−7.1079 1418 LEU257 CD2 3.4361 −12.4093 −9.1829 1419 THR258 N 3.5695−11.6026 −12.0657 1420 THR258 CA 2.6236 −12.1467 −13.048 1421 THR258 C2.3296 −13.6435 −12.8472 1422 THR258 O 1.6948 −14.3054 −13.6756 1423THR258 CB 3.0645 −11.8512 −14.4962 1424 THR258 OG1 4.2124 −12.6355−14.8271 1425 THR258 CG2 3.3907 −10.3551 −14.6699 1426 GLY259 N 2.8063−14.1799 −11.6779 1427 GLY259 CA 2.6734 −15.5863 −11.3513 1428 GLY259 C3.3389 −15.854 −10.0101 1429 GLY259 O 4.304 −15.195 −9.6162 1430 ILE260N 2.7885 −16.9007 −9.3074 1431 ILE260 CA 3.2533 −17.2739 −7.977 1432ILE260 C 2.7416 −18.6858 −7.7268 1433 ILE260 O 1.5506 −18.9623 −7.85421434 ILE260 CB 2.6632 −16.3002 −6.934 1435 ILE260 CG1 3.4254 −14.9634−6.9743 1436 ILE260 CG2 2.7358 −16.8838 −5.5089 1437 ILE260 CD1 2.5596−13.8638 −6.3365 1438 LEU272 N 7.6295 −20.2065 −5.2267 1439 LEU272 CA8.2148 −19.4305 −6.3034 1440 LEU272 C 7.2661 −18.2935 −6.6979 1441LEU272 O 6.0434 −18.3602 −6.5699 1442 LEU272 CB 8.5888 −20.3099 −7.50791443 LEU272 CG 9.6054 −21.3716 −7.0504 1444 LEU272 CD1 10.0036 −22.2306−8.2604 1445 LEU272 CD2 10.8588 −20.691 −6.4681 1446 VAL273 N 7.917−17.2324 −7.2939 1447 VAL273 CA 7.2358 −16.0184 −7.722 1448 VAL273 C7.8155 −15.6174 −9.0891 1449 VAL273 O 9.0208 −15.6826 −9.3478 1450VAL273 CB 7.4182 −14.8938 −6.6832 1451 VAL273 CG1 6.7413 −13.5994−7.1733 1452 VAL273 CG2 8.9137 −14.6382 −6.4113 1453 LYS274 N 6.862−15.1989 −10.0027 1454 LYS274 CA 7.2322 −14.6979 −11.3118 1455 LYS274 C7.1133 −13.1683 −11.2835 1456 LYS274 O 6.0599 −12.5689 −11.505 1457LYS274 CB 6.3006 −15.2891 −12.3851 1458 LYS274 CG 7.1148 −15.4963−13.6755 1459 LYS274 CD 6.3157 −14.9771 −14.8844 1460 LYS274 CE 7.0223−13.7456 −15.4826 1461 LYS274 NZ 6.9113 −12.6067 −14.5607 1462 LEU275 N8.3118 −12.5555 −10.9585 1463 LEU275 CA 8.497 −11.1202 −11.1425 1464LEU275 C 8.6314 −10.9281 −12.6662 1465 LEU275 O 9.183 −11.7675 −13.37711466 LEU275 CB 9.7478 −10.6015 −10.4053 1467 LEU275 CG 9.6452 −10.8357−8.8828 1468 LEU275 CD1 8.3234 −10.2695 −8.3293 1469 LEU275 CD2 9.7528−12.336 −8.5475 1470 ARG276 N 8.1358 −9.74 −13.1543 1471 ARG276 CA8.3421 −9.324 −14.5331 1472 ARG276 C 8.8047 −7.8552 −14.5295 1473 ARG276O 8.2417 −6.9722 −13.8836 1474 ARG276 CB 7.0444 −9.48 −15.3455 1475ARG276 CG 7.3659 −9.3005 −16.8396 1476 ARG276 CD 6.062 −9.0872 −17.62871477 ARG276 NE 6.3734 −8.4218 −18.8805 1478 ARG276 CZ 6.0922 −7.1638−19.0603 1479 ARG276 NH1 5.5179 −6.4638 −18.1263 1480 ARG276 NH2 6.3917−6.5985 −20.192 1481 ASN277 N 9.9012 −7.6359 −15.3508 1482 ASN277 CA10.3016 −6.298 −15.783 1483 ASN277 C 9.4382 −6.0331 −17.0268 1484 ASN277O 9.4635 −6.8197 −17.9793 1485 ASN277 CB 11.7935 −6.306 −16.1691 1486ASN277 CG 12.2067 −4.9569 −16.6844 1487 ASN277 OD1 12.677 −4.8526−17.8051 1488 ASN277 ND2 12.0325 −3.9052 −15.8638 1489 PRO278 N 8.6591−4.8959 −17.042 1490 PRO278 CA 7.7978 −4.6224 −18.1928 1491 PRO278 C8.507 −3.9806 −19.3944 1492 PRO278 O 7.9611 −3.9586 −20.4996 1493 PRO278CB 6.8446 −3.5703 −17.5903 1494 PRO278 CG 7.4474 −3.0849 −16.2523 1495PRO278 CD 8.643 −3.9952 −15.9106 1496 TRP279 N 9.717 −3.3532 −19.13381497 TRP279 CA 10.5405 −2.8672 −20.2519 1498 TRP279 C 11.0577 −4.086−21.0245 1499 TRP279 O 11.2024 −4.0388 −22.2464 1500 TRP279 CB 11.702−2.029 −19.6878 1501 TRP279 CG 11.2019 −0.672 −19.2777 1502 TRP279 CD19.9671 −0.1697 −19.4439 1503 TRP279 CD2 12.0185 0.3875 −18.5888 1504TRP279 NE1 9.9044 1.0428 −18.9516 1505 TRP279 CE2 11.0955 1.4041−18.4321 1506 TRP279 CE3 13.34 0.4865 −18.1559 1507 TRP279 CZ2 11.432.606 −17.8089 1508 TRP279 CZ3 13.6877 1.6954 −17.5407 1509 TRP279 CH212.7518 2.7248 −17.3612 1510 GLY280 N 11.4159 −5.1697 −20.251 1511GLY280 CA 11.5344 −6.5276 −20.7606 1512 GLY280 C 12.812 −7.2554 −20.34321513 GLY280 O 12.8765 −8.4845 −20.3353 1514 LYS281 N 13.8767 −6.4156−20.0798 1515 LYS281 CA 15.2169 −6.9354 −19.7943 1516 LYS281 C 15.1794−7.7785 −18.5004 1517 LYS281 O 14.3936 −7.5659 −17.5749 1518 LYS281 CB16.1986 −5.759 −19.6333 1519 LYS281 CG 16.1224 −4.8304 −20.8619 1520LYS281 CD 14.9288 −3.865 −20.7223 1521 LYS281 CE 14.4474 −3.4386−22.1215 1522 LYS281 NZ 13.5748 −2.261 −22.0113 1523 VAL282 N 16.105−8.809 −18.4822 1524 VAL282 CA 16.0955 −9.8258 −17.4338 1525 VAL282 C17.2642 −9.5236 −16.4779 1526 VAL282 O 17.0861 −9.1222 −15.3283 1527VAL282 CB 16.1673 −11.2512 −18.0151 1528 VAL282 CG1 16.201 −12.2832−16.8712 1529 VAL282 CG2 14.9202 −11.505 −18.8829 1530 GLU283 N 18.5204−9.7489 −17.0297 1531 GLU283 CA 19.7139 −9.1512 −16.4335 1532 GLU283 C19.9054 −9.6078 −14.9743 1533 GLU283 O 20.3128 −8.8728 −14.0762 1534GLU283 CB 19.699 −7.6156 −16.554 1535 GLU283 CG 20.3208 −7.2119 −17.90311536 GLU283 CD 19.2366 −6.8709 −18.8827 1537 GLU283 OE1 18.5651 −7.8146−19.3787 1538 GLU283 OE2 19.0641 −5.6559 −19.1671 1539 TRP284 N 19.711−10.9701 −14.8076 1540 TRP284 CA 19.6307 −11.5979 −13.4902 1541 TRP284 C20.32 −12.9674 −13.5704 1542 TRP284 O 20.2535 −13.6941 −14.5633 1543TRP284 CB 18.1888 −11.6225 −12.9414 1544 TRP284 CG 17.91 −12.8763−12.1649 1545 TRP284 CD1 18.4357 −13.2441 −10.9858 1546 TRP284 CD216.9741 −13.97 −12.5976 1547 TRP284 NE1 17.9623 −14.4142 −10.6335 1548TRP284 CE2 17.0959 −14.874 −11.5596 1549 TRP284 CE3 16.1459 −14.1717−13.7001 1550 TRP284 CZ2 16.3837 −16.0723 −11.5488 1551 TRP284 CZ315.4287 −15.3733 −13.6977 1552 TRP284 CH2 15.5441 −16.2952 −12.6471 1553LYS285 N 21.0285 −13.3247 −12.4366 1554 LYS285 CA 21.9632 −14.4504−12.445 1555 LYS285 C 21.1662 −15.7417 −12.2212 1556 LYS285 O 21.0234−16.2627 −11.1162 1557 LYS285 CB 22.9755 −14.2461 −11.3019 1558 LYS285CG 23.8492 −13.0142 −11.6019 1559 LYS285 CD 24.8293 −12.7871 −10.43671560 LYS285 CE 25.6941 −11.5475 −10.7307 1561 LYS285 NZ 26.6278 −11.3271−9.6171 1562 GLY286 N 20.6382 −16.2482 −13.3943 1563 GLY286 CA 20.0064−17.545 −13.4703 1564 GLY286 C 18.5419 −17.4677 −13.887 1565 GLY286 O18.054 −16.5184 −14.4926 1566 ASP287 N 17.8398 −18.6115 −13.5564 1567ASP287 CA 16.4395 −18.815 −13.9266 1568 ASP287 C 15.7156 −19.501−12.7544 1569 ASP287 O 14.7632 −20.2565 −12.9279 1570 ASP287 CB 16.3237−19.6363 −15.2247 1571 ASP287 CG 17.1292 −18.977 −16.3062 1572 ASP287OD1 18.2282 −19.5037 −16.6247 1573 ASP287 OD2 16.665 −17.9357 −16.84271574 TRP288 N 16.2054 −19.094 −11.5288 1575 TRP288 CA 15.7924 −19.5176−10.1793 1576 TRP288 C 17.1337 −19.6211 −9.4095 1577 TRP288 O 18.0766−20.2926 −9.8253 1578 TRP288 CB 15.2243 −20.9457 −10.3028 1579 TRP288 CG14.4441 −21.4204 −9.1144 1580 TRP288 CD1 14.2846 −20.8147 −7.9271 1581TRP288 CD2 13.6849 −22.7179 −9.0621 1582 TRP288 NE1 13.5403 −21.5602−7.1474 1583 TRP288 CE2 13.1744 −22.695 −7.779 1584 TRP288 CE3 13.4595−23.7651 −9.9543 1585 TRP288 CZ2 12.4059 −23.7469 −7.2824 1586 TRP288CZ3 12.6885 −24.8255 −9.4629 1587 TRP288 CH2 12.1814 −24.8191 −8.15531588 PRO300 N 9.0722 −32.1709 −11.9449 1589 PRO300 CA 8.8932 −32.9359−13.178 1590 PRO300 C 8.459 −32.0578 −14.3665 1591 PRO300 O 8.001−30.9196 −14.2469 1592 PRO300 CB 7.7612 −33.9139 −12.8092 1593 PRO300 CG7.3326 −33.6108 −11.3554 1594 PRO300 CD 8.2537 −32.5064 −10.7989 1595LYS301 N 8.4755 −32.7272 −15.5917 1596 LYS301 CA 8.2126 −31.983 −16.83821597 LYS301 C 6.7948 −31.3758 −16.8074 1598 LYS301 O 6.4991 −30.3471−17.4149 1599 LYS301 CB 8.3182 −32.9405 −18.0382 1600 LYS301 CG 8.4303−32.1066 −19.3278 1601 LYS301 CD 8.3916 −33.0374 −20.5528 1602 LYS301 CE8.5678 −32.1989 −21.8321 1603 LYS301 NZ 8.4029 −33.0622 −23.01 1604GLU302 N 5.8877 −32.1242 −16.0797 1605 GLU302 CA 4.4635 −31.8098−16.0248 1606 GLU302 C 4.2584 −30.4583 −15.3122 1607 GLU302 O 3.2774−29.75 −15.532 1608 GLU302 CB 3.7449 −32.9192 −15.2313 1609 GLU302 CG4.1635 −34.3169 −15.7328 1610 GLU302 CD 4.0107 −34.4234 −17.2234 1611GLU302 OE1 5.0311 −34.2282 −17.9371 1612 GLU302 OE2 2.8745 −34.7148−17.6831 1613 LYS303 N 5.1878 −30.1862 −14.3216 1614 LYS303 CA 5.2839−28.8544 −13.7347 1615 LYS303 C 6.181 −27.9679 −14.6161 1616 LYS303 O5.9116 −26.7753 −14.7922 1617 LYS303 CB 5.9465 −29.0006 −12.3508 1618LYS303 CG 5.9219 −27.6475 −11.6131 1619 LYS303 CD 6.4192 −27.8209−10.165 1620 LYS303 CE 7.945 −28.0304 −10.1462 1621 LYS303 NZ 8.4058−28.1127 −8.7527 1622 ILE304 N 7.3301 −28.5457 −15.1282 1623 ILE304 CA8.3447 −27.6959 −15.7599 1624 ILE304 C 7.7032 −26.9572 −16.9428 1625ILE304 O 7.8731 −25.7468 −17.1109 1626 ILE304 CB 9.5889 −28.5008−16.1894 1627 ILE304 CG1 10.7478 −27.5181 −16.4497 1628 ILE304 CG29.3053 −29.3183 −17.4652 1629 ILE304 CD1 12.0311 −28.283 −16.8261 1630LEU305 N 6.8641 −27.6745 −17.7763 1631 LEU305 CA 6.3499 −27.0252−18.9765 1632 LEU305 C 5.2601 −25.9522 −18.6982 1633 LEU305 O 4.7144−25.3199 −19.6105 1634 LEU305 CB 5.7725 −28.0968 −19.9225 1635 LEU305 CG6.7608 −29.2675 −20.0948 1636 LEU305 CD1 6.095 −30.3824 −20.9216 1637LEU305 CD2 8.0414 −28.7888 −20.8042 1638 LEU306 N 4.9626 −25.7193−17.3707 1639 LEU306 CA 4.19 −24.5679 −16.915 1640 LEU306 C 5.1086−23.3267 −16.7981 1641 LEU306 O 4.6468 −22.188 −16.8894 1642 LEU306 CB3.6932 −24.9148 −15.4982 1643 LEU306 CG 2.3333 −24.2509 −15.2081 1644LEU306 CD1 1.7749 −24.8107 −13.8871 1645 LEU306 CD2 2.4939 −22.7248−15.0753 1646 GLU314 N 10.4436 −13.0374 −18.0961 1647 GLU314 CA 9.8608−14.2598 −17.5136 1648 GLU314 C 10.4432 −14.4897 −16.0917 1649 GLU314 O9.7656 −14.9325 −15.1632 1650 GLU314 CB 10.1254 −15.473 −18.426 1651GLU314 CG 9.5111 −16.7608 −17.8338 1652 GLU314 CD 8.0669 −16.6372−17.4286 1653 GLU314 OE1 7.4432 −15.5794 −17.7046 1654 GLU314 OE2 7.5444−17.6074 −16.8192 1655 PHE315 N 11.8166 −14.3518 −15.9765 1656 PHE315 CA12.5183 −14.472 −14.6901 1657 PHE315 C 12.3189 −15.866 −14.031 1658PHE315 O 13.0269 −16.8309 −14.3115 1659 PHE315 CB 12.1636 −13.3303−13.7155 1660 PHE315 CG 12.6233 −11.9913 −14.2807 1661 PHE315 CD111.6801 −11.0594 −14.7211 1662 PHE315 CD2 13.9865 −11.6939 −14.3555 1663PHE315 CE1 12.0974 −9.832 −15.2422 1664 PHE315 CE2 14.4035 −10.465−14.8729 1665 PHE315 CZ 13.4607 −9.5373 −15.3237 1666 TRP316 N 11.2462−15.886 −13.1565 1667 TRP316 CA 10.9705 −16.8771 −12.1173 1668 TRP316 C12.0455 −16.8216 −11.0225 1669 TRP316 O 13.2421 −16.9797 −11.2594 1670TRP316 CB 10.7381 −18.2895 −12.6822 1671 TRP316 CG 9.2689 −18.4001−12.963 1672 TRP316 CD1 8.6753 −18.4596 −14.1651 1673 TRP316 CD2 8.1813−18.4517 −11.9242 1674 TRP316 NE1 7.3755 −18.5351 −14.0157 1675 TRP316CE2 7.0451 −18.521 −12.7075 1676 TRP316 CE3 8.1457 −18.4409 −10.53041677 TRP316 CZ2 5.7705 −18.5613 −12.1427 1678 TRP316 CZ3 6.8694 −18.4933−9.9561 1679 TRP316 CH2 5.7095 −18.5382 −10.7435 1680 MET317 N 11.5608−16.6275 −9.7416 1681 MET317 CA 12.4538 −16.6791 −8.585 1682 MET317 C11.7004 −17.2206 −7.3651 1683 MET317 O 10.5025 −17.5005 −7.3973 1684MET317 CB 13.0084 −15.2749 −8.2814 1685 MET317 CG 14.094 −14.9049 −9.3091686 MET317 SD 15.4463 −14.1 −8.4033 1687 MET317 CE 15.699 −12.6828−9.5119 1688 THR318 N 12.492 −17.4183 −6.2542 1689 THR318 CA 11.9454−17.9342 −5.0041 1690 THR318 C 11.1717 −16.829 −4.282 1691 THR318 O11.3504 −15.634 −4.5022 1692 THR318 CB 13.0703 −18.454 −4.0877 1693THR318 OG1 13.9732 −17.3917 −3.7649 1694 THR318 CG2 13.8342 −19.5924−4.7897 1695 LEU319 N 10.3978 −17.2806 −3.2302 1696 LEU319 CA 9.7894−16.3701 −2.2475 1697 LEU319 C 10.7846 −16.099 −1.0834 1698 LEU319 O10.4341 −15.8581 0.0692 1699 LEU319 CB 8.4396 −16.8786 −1.7027 1700LEU319 CG 7.7041 −15.7551 −0.9471 1701 LEU319 CD1 7.3298 −14.6195−1.9176 1702 LEU319 CD2 6.4304 −16.3237 −0.2959 1703 GLN320 N 12.099−15.9985 −1.4967 1704 GLN320 CA 13.173 −15.3505 −0.75 1705 GLN320 C13.7063 −14.1818 −1.6072 1706 GLN320 O 14.8386 −13.7152 −1.4908 1707GLN320 CB 14.2955 −16.3545 −0.4343 1708 GLN320 CG 13.8084 −17.30410.6748 1709 GLN320 CD 14.5664 −17.039 1.9426 1710 GLN320 OE1 15.1729−17.9483 2.4844 1711 GLN320 NE2 14.54 −15.7854 2.4298 1712 ASP321 N12.7319 −13.6055 −2.4064 1713 ASP321 CA 12.9573 −12.3844 −3.173 1714ASP321 C 13.1776 −11.2013 −2.2097 1715 ASP321 O 13.9278 −10.2725 −2.51091716 ASP321 CB 14.1028 −12.5416 −4.1915 1717 ASP321 CG 13.7547 −13.6243−5.1712 1718 ASP321 OD1 12.8643 −13.3811 −6.0284 1719 ASP321 OD2 14.3754−14.7177 −5.0903 1720 PHE322 N 12.475 −11.3022 −1.0119 1721 PHE322 CA12.3354 −10.223 −0.0218 1722 PHE322 C 13.6416 −9.511 0.3661 1723 PHE322O 13.64 −8.4219 0.9499 1724 PHE322 CB 11.4304 −10.6181 1.1591 1725PHE322 CG 10.0585 −11.0417 0.6353 1726 PHE322 CD1 9.2605 −11.8954 1.40141727 PHE322 CD2 9.5925 −10.5846 −0.6018 1728 PHE322 CE1 7.9945 −12.27090.9439 1729 PHE322 CE2 8.3347 −10.9736 −1.0689 1730 PHE322 CZ 7.5274−11.8041 −0.2875 1731 LYS323 N 14.7997 −10.2081 0.0947 1732 LYS323 CA16.1194 −9.6218 0.2551 1733 LYS323 C 16.1537 −8.3235 −0.5737 1734 LYS323O 16.3342 −7.2263 −0.029 1735 LYS323 CB 17.1131 −10.6095 −0.3812 1736LYS323 CG 18.4795 −9.9502 −0.6458 1737 LYS323 CD 19.1402 −10.6292−1.8584 1738 LYS323 CE 20.4432 −11.3163 −1.4133 1739 LYS323 NZ 20.5091−12.6665 −1.99 1740 THR324 N 15.8967 −8.4471 −1.9344 1741 THR324 CA15.6988 −7.2238 −2.7145 1742 THR324 C 14.1951 −6.8837 −2.728 1743 THR324O 13.3502 −7.5502 −2.1343 1744 THR324 CB 16.624 −6.0305 −2.3844 1745THR324 OG1 16.4994 −5.0168 −3.3859 1746 THR324 CG2 18.0917 −6.4955−2.3306 1747 HIS325 N 13.8893 −5.7011 −3.3613 1748 HIS325 CA 12.5701−5.0806 −3.2588 1749 HIS325 C 12.3707 −4.4943 −1.8399 1750 HIS325 O13.062 −4.7926 −0.8612 1751 HIS325 CB 11.4731 −6.1791 −3.1788 1752HIS325 CG 11.4857 −7.5352 −3.8442 1753 HIS325 ND1 11.8417 −7.758 −5.08681754 HIS325 CD2 11.1153 −8.6655 −3.2092 1755 HIS325 CE1 11.7254 −9.0281−5.3105 1756 HIS325 NE2 11.3146 −9.6062 −4.2763 1757 PHE326 N 11.3365−3.58 −1.7799 1758 PHE326 CA 11.0657 −2.793 −0.5818 1759 PHE326 C 9.5505−2.7222 −0.3591 1760 PHE326 O 9.0613 −2.9912 0.741 1761 PHE326 CB 11.643−1.3757 −0.7791 1762 PHE326 CG 10.9725 −0.3951 0.179 1763 PHE326 CD111.4 −0.3051 1.506 1764 PHE326 CD2 9.9205 0.405 −0.2744 1765 PHE326 CE110.7388 0.5484 2.393 1766 PHE326 CE2 9.2546 1.2522 0.6146 1767 PHE326 CZ9.659 1.3172 1.9505 1768 VAL327 N 8.7986 −2.2783 −1.4285 1769 VAL327 CA7.3404 −2.1816 −1.3464 1770 VAL327 C 6.7208 −3.0375 −2.455 1771 VAL327 O7.3013 −3.2595 −3.5218 1772 VAL327 CB 6.9055 −0.7062 −1.473 1773 VAL327CG1 7.4988 −0.0788 −2.749 1774 VAL327 CG2 5.3698 −0.5854 −1.5107 1775LEU328 N 5.4394 −3.474 −2.1761 1776 LEU328 CA 4.5394 −3.9376 −3.218 1777LEU328 C 3.2176 −3.1868 −3.0694 1778 LEU328 O 2.724 −2.9298 −1.9697 1779LEU328 CB 4.3597 −5.4663 −3.2082 1780 LEU328 CG 4.3722 −6.0073 −4.64951781 LEU328 CD1 4.7594 −7.4974 −4.632 1782 LEU328 CD2 2.9671 −5.859−5.2598 1783 LEU329 N 2.6001 −2.9333 −4.2765 1784 LEU329 CA 1.193−2.6048 −4.3562 1785 LEU329 C 0.5155 −3.4931 −5.4062 1786 LEU329 O1.1143 −3.9316 −6.3919 1787 LEU329 CB 0.9461 −1.1036 −4.6129 1788 LEU329CG 1.0817 −0.7385 −6.1049 1789 LEU329 CD1 0.608 0.7114 −6.3194 1790LEU329 CD2 2.55 −0.8685 −6.5509 1791 VAL330 N −0.8373 −3.6758 −5.18151792 VAL330 CA −1.6764 −4.2365 −6.2131 1793 VAL330 C −3.022 −3.5331−6.2756 1794 VAL330 O −3.6295 −3.1734 −5.2701 1795 VAL330 CB −1.869−5.7582 −6.0539 1796 VAL330 CG1 −0.5085 −6.4754 −6.1294 1797 VAL330 CG2−2.5427 −6.0744 −4.7044 1798 ILE331 N −3.4871 −3.4207 −7.5793 1799ILE331 CA −4.8617 −3.0387 −7.8651 1800 ILE331 C −5.6687 −4.3361 −7.81711801 ILE331 O −5.5087 −5.2422 −8.6338 1802 ILE331 CB −5.0263 −2.2262−9.166 1803 ILE331 CG1 −6.3283 −1.3981 −9.1359 1804 ILE331 CG2 −4.9675−3.1137 −10.4239 1805 ILE331 CD1 −7.5711 −2.308 −9.0729 1806 CYS332 N−6.5239 −4.4163 −6.736 1807 CYS332 CA −7.5339 −5.4568 −6.6303 1808CYS332 C −8.7833 −4.897 −7.3342 1809 CYS332 O −9.3973 −3.9144 −6.91991810 CYS332 CB −7.8548 −5.6814 −5.1396 1811 CYS332 SG −6.3101 −6.0673−4.2608 1812 LYS333 N −9.0627 −5.5952 −8.5015 1813 LYS333 CA −9.997−5.1317 −9.5439 1814 LYS333 C −11.4423 −5.0121 −9.0241 1815 LYS333 O−11.8644 −5.6915 −8.0867 1816 LYS333 CB −9.9678 −6.077 −10.7587 1817LYS333 CG −8.7072 −5.8054 −11.5994 1818 LYS333 CD −8.75 −6.6779 −12.86721819 LYS333 CE −7.6597 −6.2085 −13.8472 1820 LYS333 NZ −7.6919 −7.0471−15.0537 1821 LEU334 N −12.2462 −4.1357 −9.7491 1822 LEU334 CA −13.6898−4.1252 −9.5387 1823 LEU334 C −14.38 −5.1407 −10.4725 1824 LEU334 O−13.8784 −5.5852 −11.5051 1825 LEU334 CB −14.2489 −2.7054 −9.764 1826LEU334 CG −13.6956 −2.0947 −11.0682 1827 LEU334 CD1 −14.4444 −2.6689−12.2862 1828 LEU334 CD2 −13.8664 −0.5646 −11.0349 1829 THR335 N−15.6585 −5.5113 −10.0732 1830 THR335 CA −16.2374 −6.7494 −10.6099 1831THR335 C −16.3599 −6.7425 −12.1466 1832 THR335 O −16.076 −7.7687−12.7772 1833 THR335 CB −17.6125 −6.9852 −9.9543 1834 THR335 OG1 −17.482−6.932 −8.5303 1835 THR335 CG2 −18.1496 −8.3726 −10.354 1836 PRO336 N−16.8272 −5.6156 −12.7969 1837 PRO336 CA −16.8699 −5.5685 −14.2567 1838PRO336 C −15.6067 −4.9394 −14.8805 1839 PRO336 O −15.6777 −4.3094−15.9435 1840 PRO336 CB −18.0247 −4.5626 −14.4324 1841 PRO336 CG−18.0624 −3.6981 −13.1509 1842 PRO336 CD −17.2995 −4.468 −12.0539 1843GLY337 N −14.381 −5.2228 −14.289 1844 GLY337 CA −13.1765 −4.5548−14.8168 1845 GLY337 C −12.9591 −5.1022 −16.2423 1846 GLY337 O −12.8797−4.3511 −17.2155 1847 LEU338 N −12.8761 −6.4767 −16.2809 1848 LEU338 CA−12.7619 −7.2844 −17.5043 1849 LEU338 C −14.1606 −7.3168 −18.1368 1850LEU338 O −15.1946 −7.3322 −17.4563 1851 LEU338 CB −12.3491 −8.7238−17.1402 1852 LEU338 CG −11.1472 −8.7229 −16.1758 1853 LEU338 CD1−10.784 −10.1731 −15.8073 1854 LEU338 CD2 −9.9384 −8.0376 −16.8396 1855LEU339 N −14.2068 −7.4401 −19.5093 1856 LEU339 CA −15.4662 −7.5266−20.2566 1857 LEU339 C −16.0264 −8.9621 −20.1133 1858 LEU339 O −16.1698−9.7391 −21.05 1859 LEU339 CB −15.192 −7.1854 −21.7343 1860 LEU339 CG−16.5204 −7.0397 −22.5014 1861 LEU339 CD1 −17.3118 −5.8347 −21.9584 1862LEU339 CD2 −16.225 −6.8256 −23.9972 1863 LYS346 N −13.6587 −13.5204−9.0845 1864 LYS346 CA −14.0349 −12.0861 −9.0598 1865 LYS346 C −14.753−11.721 −7.7248 1866 LYS346 O −15.5288 −12.4919 −7.1579 1867 LYS346 CB−14.8475 −11.6676 −10.2994 1868 LYS346 CG −13.8718 −11.3517 −11.44871869 LYS346 CD −14.6375 −11.2352 −12.7799 1870 LYS346 CE −13.64 −10.9981−13.9304 1871 LYS346 NZ −12.7546 −12.1634 −14.0738 1872 TRP347 N−14.4396 −10.4473 −7.2103 1873 TRP347 CA −15.068 −9.9379 −5.9543 1874TRP347 C −16.5675 −9.6711 −6.1715 1875 TRP347 O −17.1483 −9.8828 −7.24031876 TRP347 CB −14.3837 −8.615 −5.5551 1877 TRP347 CG −12.8879 −8.754−5.5676 1878 TRP347 CD1 −12.0729 −8.5668 −6.6177 1879 TRP347 CD2 −12.022−9.1279 −4.3949 1880 TRP347 NE1 −10.8288 −8.7702 −6.2605 1881 TRP347 CE2−10.7582 −9.1 −4.9544 1882 TRP347 CE3 −12.2542 −9.4434 −3.0568 1883TRP347 CZ2 −9.6178 −9.3836 −4.2033 1884 TRP347 CZ3 −11.1126 −9.7177−2.2936 1885 TRP347 CH2 −9.8263 −9.6862 −2.8519 1886 THR348 N −17.2311−9.2235 −5.0483 1887 THR348 CA −18.4309 −8.3907 −5.139 1888 THR348 C−18.0481 −7.0196 −4.5307 1889 THR348 O −17.0874 −6.9101 −3.7678 1890THR348 CB −19.6085 −9.0273 −4.3772 1891 THR348 OG1 −19.828 −10.3558−4.8585 1892 THR348 CG2 −20.8846 −8.1903 −4.5859 1893 TYR349 N −18.8199−5.9544 −4.9677 1894 TYR349 CA −18.4601 −4.5467 −4.7233 1895 TYR349 C−19.692 −3.7778 −4.2256 1896 TYR349 O −20.7812 −3.8985 −4.7855 1897TYR349 CB −17.8732 −3.949 −6.0187 1898 TYR349 CG −17.9756 −2.4268 −6.0451899 TYR349 CD1 −17.1307 −1.644 −5.2536 1900 TYR349 CD2 −18.9219 −1.8173−6.8727 1901 TYR349 CE1 −17.1971 −0.2503 −5.3315 1902 TYR349 CE2−18.9874 −0.4242 −6.9511 1903 TYR349 CZ −18.1057 0.3584 −6.2015 1904TYR349 OH −18.1316 1.7425 −6.3246 1905 THR350 N −19.4222 −2.8685 −3.20061906 THR350 CA −20.5275 −2.1332 −2.5886 1907 THR350 C −20.0198 −0.8124−1.9808 1908 THR350 O −19.9987 −0.6341 −0.7551 1909 THR350 CB −21.212−3.0091 −1.5168 1910 THR350 CG1 −21.5712 −4.2818 −2.0617 1911 THR350 CG2−22.4841 −2.3087 −1.0023 1912 GLY354 N −20.2194 9.6125 1.8171 1913GLY354 CA −20.5803 10.3257 3.0207 1914 GLY354 C −19.8737 11.6442 3.22191915 GLY354 O −18.9396 12.0026 2.4973 1916 ARG355 N −20.3337 12.35954.2427 1917 ARG355 CA −19.7915 13.6605 4.6068 1918 ARG355 C −19.481613.7208 6.1058 1919 ARG355 O −20.0338 12.963 6.9176 1920 ARG355 CB−20.7626 14.7857 4.2022 1921 ARG355 CG −21.0081 14.7491 2.6824 1922ARG355 CD −22.067 15.8029 2.3093 1923 ARG355 NE −22.3912 15.6855 0.89871924 ARG355 CZ −23.607 15.4387 0.5038 1925 ARG355 NH1 −23.8574 15.3402−0.7679 1926 ARG355 NH2 −24.5773 15.2885 1.3572 1927 TRP356 N −18.60314.6557 6.4408 1928 TRP356 CA −18.2124 14.9479 7.8044 1929 TRP356 C−18.4159 16.4559 7.8518 1930 TRP356 O −17.8369 17.1917 7.0365 1931TRP356 CB −16.7627 14.5146 8.0845 1932 TRP356 CG −16.7726 13.0541 8.43471933 TRP356 CD1 −17.5396 12.4499 9.3559 1934 TRP356 CD2 −15.9162 11.98847.8061 1935 TRP356 NE1 −17.2804 11.1661 9.3854 1936 TRP356 CE2 −16.325310.8559 8.4849 1937 TRP356 CE3 −14.9384 11.9675 6.8125 1938 TRP356 CZ2−15.7751 9.6027 8.2162 1939 TRP356 CZ3 −14.3821 10.7126 6.5358 1940TRP356 CH2 −14.7886 9.5589 7.2227 1941 GLU357 N −19.3203 16.9044 8.71931942 GLU357 CA −19.606 18.3352 8.86 1943 GLU357 C −19.4842 18.827710.2964 1944 GLU357 O −19.9862 18.1955 11.2342 1945 GLU357 CB −20.987518.6842 8.2767 1946 GLU357 CG −20.9456 18.5321 6.7443 1947 GLU357 CD−21.6577 17.277 6.3304 1948 GLU357 OE1 −22.7097 17.3928 5.6473 1949GLU357 OE2 −21.1669 16.1715 6.6798 1950 LYS358 N −18.8123 19.963810.4456 1951 LYS358 CA −18.5784 20.596 11.7354 1952 LYS358 C −19.882520.9007 12.4589 1953 LYS358 O −20.7664 21.5584 11.8981 1954 LYS358 CB−17.7647 21.8918 11.564 1955 LYS358 CG −16.3456 21.5481 11.0763 1956LYS358 CD −15.5385 22.8472 10.9012 1957 LYS358 CE −14.1201 22.505210.4097 1958 LYS358 NZ −13.3483 23.7448 10.2417 1959 ARG359 N −19.98820.4295 13.7038 1960 ARG359 CA −21.1878 20.6504 14.4995 1961 ARG359 C−22.3413 19.688 14.2599 1962 ARG359 O −23.4525 19.9153 14.7234 1963ARG359 CB −21.6403 22.1238 14.5393 1964 ARG359 CG −20.4707 23.007315.0097 1965 ARG359 CD −20.7762 24.4776 14.6735 1966 ARG359 NE −19.5725.2658 14.8468 1967 ARG359 CZ −18.8966 25.6977 13.8199 1968 ARG359 NH1−17.8225 26.4039 14.0137 1969 ARG359 NH2 −19.2781 25.435 12.6043 1970SER360 N −22.4316 19.0681 13.2168 1971 SER360 CA −23.2043 17.840313.0915 1972 SER360 C −22.3742 16.6311 12.6699 1973 SER360 O −22.080315.7688 13.4894 1974 SER360 CB −24.3557 18.0463 12.0893 1975 SER360 OG−23.823 18.3466 10.7957 1976 THR361 N −21.9695 15.9826 11.893 1977THR361 CA −21.462 14.6538 11.5505 1978 THR361 C −19.9422 14.4741 11.63961979 THR361 O −19.4294 13.4251 11.272 1980 THR361 CB −21.9676 14.212810.1637 1981 THR361 OG1 −21.4778 15.1108 9.1653 1982 THR361 CG2 −23.507414.2041 10.1459 1983 ALA362 N −19.2276 15.5002 12.0997 1984 ALA362 CA−17.7763 15.4321 12.2395 1985 ALA362 C −17.4367 14.8736 13.6251 1986ALA362 O −16.9456 15.5913 14.5084 1987 ALA362 CB −17.2028 16.849512.0477 1988 GLY363 N −17.7217 13.5846 13.8023 1989 GLY363 CA −17.498112.9106 15.0732 1990 GLY363 C −16.0507 12.7893 15.4886 1991 GLY363 O−15.6953 13.0821 16.6239 1992 GLY364 N −15.2455 12.2508 14.5819 1993GLY364 CA −13.8274 12.1014 14.8131 1994 GLY364 C −13.2913 11.076615.7932 1995 GLY364 O −13.3416 11.273 17.0034 1996 GLN365 N −12.887210.5746 16.2587 1997 GLN365 CA −12.2177 9.3095 16.5323 1998 GLN365 C−12.7076 8.4801 17.7223 1999 GLN365 O −13.2636 9.0032 18.6825 2000GLN365 CB −10.6929 9.4905 16.6352 2001 GLN365 CG −10.134 9.9541 15.2782002 GLN365 CD −8.6864 10.3147 15.4393 2003 GLN365 OE1 −8.3248 11.467715.2723 2004 GLN365 NE2 −7.8401 9.3218 15.7669 2005 ARG366 N −12.5617.1283 17.6094 2006 ARG366 CA −13.1445 6.4723 18.781 2007 ARG366 C−12.8896 7.1163 20.1587 2008 ARG366 O −13.7738 7.0958 21.0315 2009ARG366 CB −12.7742 4.9759 18.7668 2010 ARG366 CG −11.2472 4.7882 18.84462011 ARG366 CD −10.8011 4.8256 20.3175 2012 ARG366 NE −9.3898 5.154720.3869 2013 ARG366 CZ −8.5786 4.479 21.1478 2014 ARG366 NH1 −9.00323.4848 21.8713 2015 ARG366 NH2 −7.3211 4.8053 21.1864 2016 PHE373 N−17.8554 11.3119 17.8872 2017 PHE373 CA −17.7516 9.8813 17.6262 2018PHE373 C −19.0308 9.2625 17.0505 2019 PHE373 O −19.2568 8.0759 17.21722020 PHE373 CB −16.9451 9.0389 18.6357 2021 PHE373 CG −16.324 7.847617.9125 2022 PHE373 CD1 −15.6237 8.036 16.7175 2023 PHE373 CD2 −16.46286.5632 18.4445 2024 PHE373 CE1 −15.099 6.9361 16.0341 2025 PHE373 CE2−15.9354 5.4632 17.7631 2026 PHE373 CZ −15.2611 5.6491 16.5535 2027TRP374 N −19.9905 8.9731 17.9207 2028 TRP374 CA −21.2595 8.3699 17.52522029 TRP374 C −22.0245 9.1861 16.4926 2030 TRP374 O −23.0368 8.725315.9713 2031 TRP374 CB −22.1189 8.2192 18.7948 2032 TRP374 CG −22.33289.5651 19.4249 2033 TRP374 CD1 −21.6289 10.1063 20.4312 2034 TRP374 CD2−23.3884 10.5621 19.0313 2035 TRP374 NE1 −22.1033 11.2936 20.7179 2036TRP374 CE2 −23.1461 11.5948 19.9167 2037 TRP374 CE3 −24.4029 10.599918.0759 2038 TRP374 CZ2 −23.9217 12.7536 19.9176 2039 TRP374 CZ3−25.1882 11.759 18.072 2040 TRP374 CH2 −24.9569 12.8067 18.9757 2041LYS375 N −21.5275 10.3856 16.1942 2042 LYS375 CA −22.1675 11.278915.2353 2043 LYS375 C −21.7208 11.1765 13.7615 2044 LYS375 O −22.319511.8173 12.8934 2045 LYS375 CB −21.7945 12.7149 15.6462 2046 LYS375 CG−22.7664 13.2608 16.7055 2047 LYS375 CD −22.5243 14.7753 16.843 2048LYS375 CE −23.3317 15.3345 18.0282 2049 LYS375 NZ −22.4946 15.329319.2364 2050 ASN376 N −20.6742 10.3986 13.4772 2051 ASN376 CA −20.217810.2068 12.0918 2052 ASN376 C −21.271 9.3361 11.4082 2053 ASN376 O−22.0253 8.6268 12.0802 2054 ASN376 CB −18.8283 9.544 12.0305 2055ASN376 CG −17.8494 10.3106 12.8735 2056 ASN376 OD1 −17.0336 11.046912.3438 2057 ASN376 ND2 −17.9206 10.135 14.2054 2058 PRO377 N −21.33619.3671 10.0661 2059 PRO377 CA −22.3291 8.5243 9.388 2060 PRO377 C−22.057 7.0623 9.8079 2061 PRO377 O −20.9748 6.7568 10.3328 2062 PRO377CB −21.9585 8.734 7.9066 2063 PRO377 CG −20.8463 9.8082 7.8403 2064PRO377 CD −20.4446 10.1914 9.2799 2065 GLN378 N −23.0285 6.1737 9.60472066 GLN378 CA −22.871 4.7658 9.9888 2067 GLN378 C −23.3197 3.8135 8.8882068 GLN378 O −24.2184 4.1278 8.1139 2069 GLN378 CB −23.5706 4.441911.321 2070 GLN378 CG −22.6472 4.8381 12.4894 2071 GLN378 CD −23.05596.1428 13.1145 2072 GLN378 OE1 −24.035 6.7518 12.7144 2073 GLN378 NE2−22.2862 6.5883 14.1234 2074 PHE379 N −22.6924 2.6423 8.8249 2075 PHE379CA −23.0117 1.663 7.7884 2076 PHE379 C −23.1047 0.251 8.3362 2077 PHE379O −22.7774 0.007 9.4898 2078 PHE379 CB −21.964 1.751 6.6627 2079 PHE379CG −21.9246 3.1751 6.118 2080 PHE379 CD1 −20.9971 4.0886 6.6254 2081PHE379 CD2 −22.8168 3.5682 5.1172 2082 PHE379 CE1 −20.9784 5.4026 6.15072083 PHE379 CE2 −22.7849 4.8767 4.6284 2084 PHE379 CZ −21.8707 5.7965.1499 2085 LEU380 N −23.5633 −0.6734 7.5016 2086 LEU380 CA −23.6919−2.0542 7.9269 2087 LEU380 C −23.0878 −3.0227 6.9376 2088 LEU380 O−23.4873 −3.0586 5.7696 2089 LEU380 CB −25.1596 −2.4145 8.2242 2090LEU380 CG −25.2241 −3.7603 8.9722 2091 LEU380 CD1 −24.5552 −3.624110.3532 2092 LEU380 CD2 −26.6953 −4.1756 9.1552 2093 LEU381 N −22.1021−3.782 7.3987 2094 LEU381 CA −21.5005 −4.808 6.5733 2095 LEU381 C−21.4522 −6.0765 7.4047 2096 LEU381 O −21.1832 −6.0301 8.6037 2097LEU381 CB −20.0819 −4.4019 6.1319 2098 LEU381 CG −20.1459 −3.1452 5.24332099 LEU381 CD1 −18.7168 −2.6707 4.9222 2100 LEU381 CD2 −20.8874 −3.46413.931 2101 LEU399 N −11.2797 −5.8873 3.0256 2102 LEU399 CA −11.0656−5.2862 1.7318 2103 LEU399 C −11.7767 −3.9423 1.8086 2104 LEU399 O−13.0068 −3.8811 1.7749 2105 LEU399 CB −11.6931 −6.1679 0.6362 2106LEU399 CG −10.8241 −6.0892 −0.6329 2107 LEU399 CD1 −9.5321 −6.9031−0.4288 2108 LEU399 CD2 −11.6064 −6.6564 −1.8309 2109 VAL400 N −11.7072−3.2732 2.9534 2110 VAL400 CA −12.3614 −1.9744 3.0903 2111 VAL400 C−11.4375 −0.7941 2.7835 2112 VAL400 O −10.3307 −0.6825 3.325 2113 VAL400CB −13.1588 −1.7882 4.3968 2114 VAL400 CG1 −13.8429 −0.4074 4.412 2115VAL400 CG2 −14.2414 −2.8795 4.4962 2116 SER401 N −11.9045 0.062 1.87492117 SER401 CA −11.1554 1.2383 1.489 2118 SER401 C −11.992 2.4792 1.71042119 SER401 O −13.1049 2.5887 1.1756 2120 SER401 CB −10.6612 1.13330.0332 2121 SER401 OG −11.7598 0.8699 −0.8441 2122 LEU402 N −11.48233.3815 2.5481 2123 LEU402 CA −12.1437 4.6486 2.8585 2124 LEU402 C−11.2989 5.7178 2.21 2125 LEU402 O −10.1325 5.8508 2.551 2126 LEU402 CB−12.0995 4.7962 4.3943 2127 LEU402 CG −12.4102 6.2361 4.8492 2128 LEU402CD1 −13.8656 6.5997 4.5007 2129 LEU402 CD2 −12.2011 6.3444 6.3717 2130LEU403 N −11.8967 6.5044 1.3209 2131 LEU403 CA −11.1725 7.5474 0.60332132 LEU403 C −11.7708 8.9329 0.8565 2133 LEU403 O −12.9664 9.06961.0485 2134 LEU403 CB −11.117 7.218 −0.9012 2135 LEU403 CG −9.85897.8275 −1.5495 2136 LEU403 CD1 −9.5662 7.0971 −2.873 2137 LEU403 CD2−10.0937 9.3191 −1.8493 2138 GLN404 N −10.9207 9.9471 0.9415 2139 GLN404CA −11.3855 11.3051 1.1758 2140 GLN404 C −11.3453 12.1121 −0.1269 2141GLN404 O −10.3735 12.0411 −0.8945 2142 GLN404 CB −10.5112 12.0111 2.2282143 GLN404 CG −9.0183 11.8766 1.864 2144 GLN404 CD −8.1937 12.98992.4457 2145 GLN404 OE1 −8.7227 13.9938 2.8945 2146 GLN404 NE2 −6.861312.8104 2.4463 2147 LYS405 N −12.4222 12.8483 −0.3878 2148 LYS405 CA−12.518 13.6554 −1.5974 2149 LYS405 C −11.798 14.9875 −1.4256 2150LYS405 O −11.5074 15.4054 −0.2997 2151 LYS405 CB −13.985 13.8319 −2.02912152 LYS405 CG −14.4537 12.5454 −2.7338 2153 LYS405 CD −15.9403 12.677−3.112 2154 LYS405 CE −16.417 11.381 −3.7939 2155 LYS405 NZ −16.186111.4678 −5.243 2156 PRO406 N −11.5033 15.637 −2.552 2157 PRO406 CA−10.8137 16.9279 −2.5722 2158 PRO406 C −11.7236 18.0933 −2.1786 2159PRO406 O −12.9466 18.0443 −2.3818 2160 PRO406 CB −10.4066 17.0777−4.0502 2161 PRO406 CG −11.1618 15.9903 −4.8462 2162 PRO406 CD −11.842515.0486 −3.833 2163 ARG407 N −11.1004 19.1521 −1.6506 2164 ARG407 CA−11.8132 20.3502 −1.2146 2165 ARG407 C −10.9625 21.6281 −1.3154 2166ARG407 O −10.4741 22.1387 −0.3026 2167 ARG407 CB −12.2986 20.1195 0.22852168 ARG407 CG −13.75 20.6143 0.3605 2169 ARG407 CD −14.5797 19.54141.0886 2170 ARG407 NE −14.806 18.4195 0.1955 2171 ARG407 CZ −14.129617.3139 0.3173 2172 ARG407 NH1 −13.2197 17.1765 1.2366 2173 ARG407 NH2−14.3691 16.33 −0.4971 2174 HIS408 N −11.0114 22.2129 −2.2375 2175HIS408 CA −9.8249 23.0234 −2.5578 2176 HIS408 C −9.1699 23.7237 −1.36492177 HIS408 O −8.391 24.3342 −1.9958 2178 HIS408 CB −10.163 23.9089−3.7771 2179 HIS408 CG −9.6012 23.2388 −4.9957 2180 HIS408 ND1 −9.911222.0018 −5.3088 2181 HIS408 CD2 −8.7462 23.8055 −5.8684 2182 HIS408 CE1−9.2706 21.7087 −6.3952 2183 HIS408 NE2 −8.5872 22.6943 −6.7667 2184ARG409 N −8.5536 23.516 −0.9303 2185 ARG409 CA −7.3223 23.5699 −0.14012186 ARG409 C −6.0983 22.8344 −0.7238 2187 ARG409 O −4.9844 23.3773−0.7235 2188 ARG409 CB −7.6311 22.9841 1.2506 2189 ARG409 CG −6.510223.363 2.2356 2190 ARG409 CD −6.7839 22.7001 3.5974 2191 ARG409 NE−5.682 22.9836 4.4984 2192 ARG409 CZ −4.9048 22.0328 4.9303 2193 ARG409NH1 −5.0867 20.7964 4.5691 2194 ARG409 NH2 −3.9285 22.3251 5.7372 2195CYS410 N −6.305 21.6096 −1.211 2196 CYS410 CA −5.2291 20.8006 −1.78782197 CYS410 C −4.7437 21.339 −3.1387 2198 CYS410 O −5.4734 21.2892−4.1333 2199 CYS410 CB −5.7276 19.3545 −1.9733 2200 CYS410 SG −6.139918.6764 −0.3384 2201 ARG411 N −3.5047 21.8272 −3.1759 2202 ARG411 CA−2.9217 22.3682 −4.408 2203 ARG411 C −2.3706 21.2465 −5.2891 2204 ARG411O −2.334 21.365 −6.521 2205 ARG411 CB −1.8105 23.366 −4.0351 2206 ARG411CG −2.2844 24.798 −4.3547 2207 ARG411 CD −3.3099 25.296 −3.315 2208ARG411 NE −4.463 24.4149 −3.2693 2209 ARG411 CZ −5.5445 24.6704 −3.9462210 ARG411 NH1 −5.6424 25.7334 −4.689 2211 ARG411 NH2 −6.5441 23.8429−3.8791 2212 LYS412 N −2.0034 20.155 −4.6818 2213 LYS412 CA −1.347718.9915 −5.2731 2214 LYS412 C −2.2703 17.8226 −5.6321 2215 LYS412 O−1.8677 16.889 −6.3285 2216 LYS412 CB 0.1347 18.7248 −4.9605 2217 LYS412CG 0.9794 19.5622 −5.9387 2218 LYS412 CD 2.4766 19.3549 −5.6505 2219LYS412 CE 3.3033 20.2142 −6.6246 2220 LYS412 NZ 4.6206 19.5956 −6.82992221 ILE419 N −8.6232 9.5358 6.8454 2222 ILE419 CA −8.7953 9.3093 8.28322223 ILE419 C −8.8493 7.8239 8.5702 2224 ILE419 O −9.1131 7.0324 7.67082225 ILE419 CB −10.0831 9.9968 8.7935 2226 ILE419 CG1 −11.3607 9.37618.1849 2227 ILE419 CG2 −10.0433 11.5167 8.5429 2228 ILE419 CD1 −11.41389.5647 6.6559 2229 GLY420 N −9.4897 7.282 9.2986 2230 GLY420 CA −9.56575.8953 9.7181 2231 GLY420 C −11.0225 5.5333 9.9443 2232 GLY420 O−11.8765 6.3862 9.9751 2233 PHE421 N −11.2936 4.2932 10.0838 2234 PHE421CA −12.6684 3.8445 10.3389 2235 PHE421 C −12.6373 2.6376 11.2691 2236PHE421 O −11.9166 1.7455 11.091 2237 PHE421 CB −13.5136 3.6209 9.06762238 PHE421 CG −12.8762 2.5907 8.1403 2239 PHE421 CD1 −12.9824 1.22658.4257 2240 PHE421 CD2 −12.1885 3.014 6.9999 2241 PHE421 CE1 −12.38160.2863 7.5846 2242 PHE421 CE2 −11.6042 2.0735 6.1476 2243 PHE421 CZ−11.6955 0.7108 6.444 2244 TYR422 N −13.3419 2.6273 12.2494 2245 TYR422CA −13.4014 1.4742 13.1552 2246 TYR422 C −14.673 0.7253 12.8963 2247TYR422 O −15.7005 1.3588 12.6603 2248 TYR422 CB −13.4225 1.896 14.63862249 TYR422 CG −12.5988 3.1533 14.9 2250 TYR422 CD1 −13.133 4.411614.6103 2251 TYR422 CD2 −11.3139 3.0477 15.4382 2252 TYR422 CE1 −12.39585.5657 14.8872 2253 TYR422 CE2 −10.5773 4.2018 15.7175 2254 TYR422 CZ−11.1209 5.4606 15.449 2255 TYR422 OH −10.3932 6.6081 15.7404 2256LEU423 N −14.6042 −0.5562 12.874 2257 LEU423 CA −15.8179 −1.3236 12.67192258 LEU423 C −16.1001 −2.2128 13.8968 2259 LEU423 O −15.2057 −2.895114.3503 2260 LEU423 CB −15.6594 −2.222 11.4302 2261 LEU423 CG −17.0006−2.3612 10.6831 2262 LEU423 CD1 −16.8092 −3.278 9.4606 2263 LEU423 CD2−18.0756 −2.9663 11.6055 2264 TYR424 N −17.3096 −2.1397 14.4203 2265TYR424 CA −17.6813 −3.0385 15.5276 2266 TYR424 C −18.874 −3.8529 15.01552267 TYR424 O −19.8515 −3.2687 14.737 2268 TYR424 CB −18.0616 −2.193916.7602 2269 TYR424 CG −17.0839 −1.0307 16.913 2270 TYR424 CD1 −17.37660.2036 16.3266 2271 TYR424 CD2 −15.8977 −1.1963 17.6328 2272 TYR424 CE1−16.4886 1.2738 16.4637 2273 TYR424 CE2 −15.0125 −0.1243 17.7756 2274TYR424 CZ −15.3148 1.115 17.2048 2275 TYR424 OH −14.4498 2.1898 17.37592276 SER18 N −29.219 12.2461 −11.1118 2277 SER18 CA −28.5696 11.9899−12.3855 2278 SER18 C −27.2585 11.2838 −12.1555 2279 SER18 O −27.002610.8074 −11.0608 2280 SER18 CB −28.3112 13.3475 −13.0696 2281 SER18 OG−27.4242 14.143 −12.2771 2282 ARG19 N −26.4112 11.2244 −13.2015 2283ARG19 CA −25.1037 10.6172 −13.0182 2284 ARG19 C −24.0502 11.6653−12.7938 2285 ARG19 O −24.3376 12.8489 −12.8706 2286 ARG19 CB −24.72959.8228 −14.2824 2287 ARG19 CG −25.4163 8.4463 −14.2455 2288 ARG19 CD−24.9216 7.6042 −15.4352 2289 ARG19 NE −25.384 6.2371 −15.2815 2290ARG19 CZ −26.1887 5.697 −16.1504 2291 ARG19 NH1 −26.5806 4.4696 −15.97892292 ARG19 NH2 −26.6077 6.3625 −17.1867 2293 ARG20 N −22.8103 11.2111−12.5208 2294 ARG20 CA −21.7107 12.1564 −12.4228 2295 ARG20 C −21.298312.5578 −13.8122 2296 ARG20 O −20.9314 13.6994 −14.038 2297 ARG20 CB−20.5429 11.5144 −11.6414 2298 ARG20 CG −19.6366 10.6438 −12.5364 2299ARG20 CD −20.4078 9.4148 −13.0514 2300 ARG20 NE −20.1989 8.3033 −12.14362301 ARG20 CZ −19.6059 7.215 −12.5401 2302 ARG20 NH1 −19.4299 6.2426−11.6958 2303 ARG20 NH2 −19.1839 7.0845 −13.764 2304 ALA21 N −21.382911.5916 −14.7481 2305 ALA21 CA −21.0788 11.8988 −16.1328 2306 ALA21 C−22.0716 12.8888 −16.6703 2307 ALA21 O −21.7518 13.5873 −17.6179 2308ALA21 CB −21.1798 10.5933 −16.9424 2309 SER22 N −23.2695 12.9551−16.0522 2310 SER22 CA −24.2611 13.9176 −16.5033 2311 SER22 C −23.682215.3066 −16.5316 2312 SER22 O −23.6389 15.8754 −17.6094 2313 SER22 CB−25.5205 13.8526 −15.6185 2314 SER22 OG −26.0658 12.5317 −15.6598 2315PRO23 N −23.2183 15.8635 −15.3902 2316 PRO23 CA −22.571 17.1607 −15.41632317 PRO23 C −21.2635 17.0693 −16.1522 2318 PRO23 O −20.8147 18.0635−16.6991 2319 PRO23 CB −22.2752 17.4063 −13.9234 2320 PRO23 CG −22.611916.1125 −13.1508 2321 PRO23 CD −23.3322 15.1657 −14.1283 2322 GLN24 N−20.6603 15.8634 −16.1775 2323 GLN24 CA −19.433 15.6899 −16.9365 2324GLN24 C −19.6586 16.0109 −18.3889 2325 GLN24 O −18.7296 16.44 −19.05342326 GLN24 CB −18.9822 14.2236 −16.7999 2327 GLN24 CG −17.8675 14.121−15.7416 2328 GLN24 CD −18.1193 12.964 −14.8176 2329 GLN24 OE1 −18.180413.1523 −13.6139 2330 GLN24 NE2 −18.2685 11.7477 −15.3715 2331 GLN25 N−20.9014 15.8186 −18.8769 2332 GLN25 CA −21.1908 16.151 −20.262 2333GLN25 C −20.9543 17.6163 −20.5161 2334 GLN25 O −20.1181 17.9192 −21.3522335 GLN25 CB −22.6443 15.7729 −20.6035 2336 GLN25 CG −22.7804 14.244−20.7335 2337 GLN25 CD −23.9393 13.7462 −19.9172 2338 GLN25 OE1 −23.804212.7627 −19.2084 2339 GLN25 NE2 −25.0992 14.4217 −20.0082 2340 PRO26 N−21.6509 18.5378 −19.8106 2341 PRO26 CA −21.3793 19.9524 −19.9756 2342PRO26 C −19.9355 20.2396 −19.6769 2343 PRO26 O −19.3935 21.1953 −20.20822344 PRO26 CB −22.2515 20.5874 −18.8742 2345 PRO26 CG −23.0722 19.4644−18.2035 2346 PRO26 CD −22.6617 18.1328 −18.8588 2347 GLN27 N −19.312819.3876 −18.838 2348 GLN27 CA −17.8868 19.5279 −18.6092 2349 GLN27 C−17.1497 18.9872 −19.8049 2350 GLN27 O −16.4509 17.9906 −19.7081 2351GLN27 CB −17.5082 18.7621 −17.3247 2352 GLN27 CG −18.2592 19.3411 −16.112353 GLN27 CD −17.7275 20.7009 −15.7592 2354 GLN27 OE1 −17.1188 20.8629−14.7145 2355 GLN27 NE2 −17.9586 21.6981 −16.6322 2356 TYR84 N −1.97564.3397 −31.0125 2357 TYR84 CA −1.4483 3.2203 −30.2523 2358 TYR84 C−1.5153 2.0149 −31.1458 2359 TYR84 O −2.2802 1.0942 −30.904 2360 TYR84CB −2.2419 3.0353 −28.9389 2361 TYR84 CG −1.6037 2.0109 −27.9974 2362TYR84 CD1 −1.8801 2.0732 −26.6288 2363 TYR84 CD2 −0.7562 1.0093 −28.48022364 TYR84 CE1 −1.3609 1.1086 −25.7608 2365 TYR84 CE2 −0.2246 0.0498−27.6157 2366 TYR84 CZ −0.5326 0.0971 −26.2544 2367 TYR84 OH −0.0121−0.8597 −25.3925 2368 PHE85 N −0.6833 2.0379 −32.2039 2369 PHE85 CA−0.6605 0.8992 −33.1028 2370 PHE85 C −0.1316 −0.33 −32.4212 2371 PHE85 O0.8114 −0.2439 −31.6511 2372 PHE85 CB 0.0502 1.1935 −34.4406 2373 PHE85CG 1.4461 1.7991 −34.2859 2374 PHE85 CD1 2.2748 1.8618 −35.409 2375PHE85 CD2 1.908 2.2915 −33.0616 2376 PHE85 CE1 3.5469 2.4316 −35.31732377 PHE85 CE2 3.1787 2.8638 −32.9666 2378 PHE85 CZ 4.0037 2.923−34.0921 2379 ALA86 N −0.7817 −1.4764 −32.7076 2380 ALA86 CA −0.3826−2.7278 −32.0819 2381 ALA86 C −0.6149 −2.7582 −30.596 2382 ALA86 O−0.7942 −1.7247 −29.9715 2383 ALA86 CB 1.0752 −3.1029 −32.4117 2384LYS87 N −0.6034 −3.9801 −30.027 2385 LYS87 CA −0.671 −4.0869 −28.57992386 LYS87 C 0.6572 −3.6505 −28.0358 2387 LYS87 O 0.704 −2.8939 −27.07992388 LYS87 CB −0.8607 −5.5636 −28.1861 2389 LYS87 CG −2.293 −6.028−28.5039 2390 LYS87 CD −2.4255 −7.5123 −28.1168 2391 LYS87 CE −3.8179−8.0329 −28.5167 2392 LYS87 NZ −3.8456 −9.4955 −28.374 2393 ALA88 N1.7382 −4.1405 −28.6778 2394 ALA88 CA 3.0742 −3.7712 −28.2453 2395 ALA88C 3.3688 −4.3514 −26.8917 2396 ALA88 O 3.2386 −3.6715 −25.8867 2397ALA88 CB 3.2728 −2.2438 −28.2909 2398 LYS89 N 3.7874 −5.6324 −26.8842399 LYS89 CA 4.1863 −6.2498 −25.6303 2400 LYS89 C 5.3313 −5.4685−25.0515 2401 LYS89 O 5.3175 −5.1573 −23.8715 2402 LYS89 CB 4.6519−7.6849 −25.942 2403 LYS89 CG 5.1193 −8.3944 −24.6573 2404 LYS89 CD5.6894 −9.7741 −25.0329 2405 LYS89 CE 6.2991 −10.443 −23.7881 2406 LYS89NZ 7.6382 −10.9439 −24.1273 2407 ARG90 N 6.3187 −5.1388 −25.9065 2408ARG90 CA 7.4058 −4.306 −25.43 2409 ARG90 C 6.9689 −2.8753 −25.2833 2410ARG90 O 5.8984 −2.4983 −25.7329 2411 ARG90 CB 8.6259 −4.4176 −26.36272412 ARG90 CG 9.4794 −5.6297 −25.944 2413 ARG90 CD 10.1419 −5.3493−24.582 2414 ARG90 NE 10.8092 −6.5457 −24.1021 2415 ARG90 CZ 12.0871−6.5553 −23.854 2416 ARG90 NH1 12.8194 −5.4944 −24.0281 2417 ARG90 NH212.6424 −7.6489 −23.4233 2418 LEU91 N 7.8313 −2.0836 −24.6216 2419 LEU91CA 7.4596 −0.7216 −24.2832 2420 LEU91 C 8.727 0.0817 −24.2932 2421 LEU91O 9.7323 −0.4426 −23.8379 2422 LEU91 CB 7.0082 −0.8199 −22.8106 2423LEU91 CG 6.2327 0.4149 −22.3125 2424 LEU91 CD1 5.823 0.1552 −20.851 2425LEU91 CD2 7.1128 1.6773 −22.353 2426 HIS113 N −6.4353 −10.6403 −22.92622427 HIS113 CA −7.4859 −10.5959 −21.9252 2428 HIS113 C −7.1733 −9.4431−21.0038 2429 HIS113 O −6.1033 −8.8622 −21.1001 2430 HIS113 CB −7.5034−11.9014 −21.1055 2431 HIS113 CG −8.8663 −12.5314 −21.1377 2432 HIS113ND1 −9.3863 −13.1002 −20.0746 2433 HIS113 CD2 −9.6792 −12.5864 −22.21072434 HIS113 CE1 −10.5551 −13.5481 −20.4069 2435 HIS113 NE2 −10.784−13.2858 −21.6127 2436 GLN114 N −8.1139 −9.1025 −20.0995 2437 GLN114 CA−7.8701 −8.0172 −19.1592 2438 GLN114 C −7.7198 −6.6951 −19.8615 2439GLN114 O −6.6331 −6.1411 −19.9172 2440 GLN114 CB −6.6818 −8.3332−18.2286 2441 GLN114 CG −7.1246 −9.3496 −17.16 2442 GLN114 CD −5.9534−9.736 −16.3046 2443 GLN114 OE1 −5.5315 −10.8802 −16.3337 2444 GLN114NE2 −5.4155 −8.779 −15.5274 2445 ASP115 N −8.8445 −6.1879 −20.4018 2446ASP115 CA −8.7767 −4.9376 −21.1382 2447 ASP115 C −9.0522 −3.7711−20.2284 2448 ASP115 O −10.0728 −3.1112 −20.3435 2449 ASP115 CB −9.7649−5.0067 −22.3186 2450 ASP115 CG −9.3264 −6.0903 −23.2611 2451 ASP115 OD1−9.6038 −7.2836 −22.964 2452 ASP115 OD2 −8.7116 −5.7495 −24.3065 2453THR128 N −5.2098 9.6232 −37.965 2454 THR128 CA −5.5784 9.5422 −39.36652455 THR128 C −6.9869 10.0144 −39.5951 2456 THR128 O −7.1718 11.0374−40.2344 2457 THR128 CB −5.3515 8.1277 −39.9358 2458 THR128 OG1 −6.13297.164 −39.2253 2459 THR128 CG2 −3.86 7.7591 −39.8286 2460 GLU129 N−7.9852 9.2654 −39.0864 2461 GLU129 CA −9.3583 9.6237 −39.4022 2462GLU129 C −9.9398 10.5726 −38.3948 2463 GLU129 O −9.6855 10.4263 −37.20972464 GLU129 CB −10.2665 8.3833 −39.5128 2465 GLU129 CG −9.5043 7.2117−40.1622 2466 GLU129 CD −9.12 6.2042 −39.116 2467 GLU129 OE1 −9.31154.9868 −39.3768 2468 GLU129 OE2 −8.6361 6.6199 −38.0287 2469 LYS130 N−10.7255 11.5496 −38.8967 2470 LYS130 CA −11.3809 12.5048 −38.0141 2471LYS130 C −10.4409 13.0512 −36.976 2472 LYS130 O −10.7529 13.0495−35.7953 2473 LYS130 CB −12.6455 11.888 −37.3856 2474 LYS130 CG −13.819612.0229 −38.3713 2475 LYS130 CD −15.1017 11.4827 −37.7127 2476 LYS130 CE−16.286 11.6603 −38.6803 2477 LYS130 NZ −17.5138 11.1586 −38.0469 2478VAL156 N 7.1495 20.8156 −25.2479 2479 VAL156 CA 8.4489 20.3852 −25.722480 VAL156 C 9.3571 21.5798 −25.7576 2481 VAL156 O 9.3263 22.352−26.7025 2482 VAL156 CB 8.3542 19.5969 −27.0437 2483 VAL156 CG1 7.352718.4379 −26.8884 2484 VAL156 CG2 7.9085 20.4856 −28.2191 2485 ASN157 N10.171 21.7158 −24.6912 2486 ASN157 CA 11.0863 22.8404 −24.6216 2487ASN157 C 11.9997 22.83 −25.8116 2488 ASN157 O 12.9363 22.049 −25.86582489 ASN157 CB 11.8895 22.7676 −23.3092 2490 ASN157 CG 12.0553 24.1436−22.7314 2491 ASN157 OD1 11.62 24.3951 −21.62 2492 ASN157 ND2 12.695125.0539 −23.4855 2493 GLU158 N 11.693 23.7209 −26.7747 2494 GLU158 CA12.4778 23.7633 −27.9961 2495 GLU158 C 12.3657 22.4554 −28.7313 2496GLU158 O 13.3724 21.8341 −29.0331 2497 GLU158 CB 13.9391 24.1693−27.7137 2498 GLU158 CG 13.9672 25.4183 −26.8115 2499 GLU158 CD 14.827125.1819 −25.6025 2500 GLU158 OE1 14.7949 24.0494 −25.0491 2501 GLU158OE2 15.5511 26.1332 −25.2057 2502 ALA159 N 11.111 22.0446 −29.0137 2503ALA159 CA 10.8855 20.7997 −29.7342 2504 ALA159 C 11.0809 19.5888−28.8664 2505 ALA159 O 10.2895 18.6639 −28.9558 2506 ALA159 CB 11.736420.701 −31.0156 2507 GLY160 N 12.1317 19.5892 −28.0226 2508 GLY160 CA12.365 18.4278 −27.1826 2509 GLY160 C 11.2963 18.3167 −26.1341 2510GLY160 O 11.1203 19.2402 −25.3565 2511 GLN161 N 10.5875 17.1693 −26.12092512 GLN161 CA 9.5227 16.9887 −25.1454 2513 GLN161 C 10.032 17.0777−23.7338 2514 GLN161 O 11.2329 17.0787 −23.5131 2515 GLN161 CB 8.842615.6214 −25.3495 2516 GLN161 CG 8.2536 15.5302 −26.7698 2517 GLN161 CD7.5633 14.2099 −26.952 2518 GLN161 OE1 6.3575 14.1746 −27.1342 2519GLN161 NE2 8.3294 13.105 −26.907 2520 GLN196 N 1.0418 9.8707 −5.495 2521GLN196 CA 1.5364 8.5086 −5.399 2522 GLN196 C 1.1702 7.6878 −6.6023 2523GLN196 O 0.4086 8.1229 −7.4515 2524 GLN196 CB 0.8757 7.8228 −4.1892 2525GLN196 CG 1.6729 8.1168 −2.9065 2526 GLN196 CD 0.849 7.7169 −1.7172 2527GLN196 OE1 0.5058 8.5584 −0.9035 2528 GLN196 NE2 0.5223 6.4166 −1.60582529 VAL197 N 1.7343 6.4652 −6.6453 2530 VAL197 CA 1.3872 5.552 −7.71922531 VAL197 C 0.0529 4.9215 −7.4393 2532 VAL197 O −0.7245 4.7517 −8.36272533 VAL197 CB 2.4636 4.454 −7.8415 2534 VAL197 CG1 3.7945 5.0789−8.2996 2535 VAL197 CG2 2.6678 3.7271 −6.4975 2536 SER198 N −0.20954.5813 −6.1611 2537 SER198 CA −1.4755 3.9503 −5.8222 2538 SER198 C−2.6401 4.8349 −6.17 2539 SER198 O −3.6232 4.3419 −6.6982 2540 SER198 CB−1.4875 3.6546 −4.31 2541 SER198 OG −1.2465 4.856 −3.5706 2542 THR209 N−6.394 −0.2325 −4.529 2543 THR209 CA −4.9737 −0.4368 −4.745 2544 THR209C −4.3568 −0.8171 −3.4284 2545 THR209 O −3.5231 −0.1004 −2.8972 2546THR209 CB −4.3323 0.7936 −5.4215 2547 THR209 OG1 −4.9557 1.007 −6.69152548 THR209 CG2 −2.8285 0.5495 −5.6524 2549 MET210 N −4.7905 −1.9815−2.9044 2550 MET210 CA −4.2352 −2.4464 −1.6446 2551 MET210 C −2.7662−2.7233 −1.7981 2552 MET210 O −2.3122 −2.9945 −2.8983 2553 MET210 CB−4.9699 −3.7096 −1.1552 2554 MET210 CG −6.4845 −3.437 −1.0776 2555MET210 SD −7.1137 −4.1282 0.4811 2556 MET210 CE −8.7771 −3.3982 0.43492557 THR211 N −2.0041 −2.6325 −0.692 2558 THR211 CA −0.5678 −2.7725−0.8487 2559 THR211 C 0.0522 −3.5941 0.2431 2560 THR211 O −0.4589−3.6484 1.3501 2561 THR211 CB 0.1235 −1.3971 −0.9424 2562 THR211 OG1−0.0089 −0.6997 0.2994 2563 THR211 CG2 −0.5138 −0.5555 −2.064 2564ILE212 N 1.1822 −4.2356 −0.1071 2565 ILE212 CA 1.9143 −5.0079 0.87862566 ILE212 C 3.3518 −4.9831 0.448 2567 ILE212 O 3.6862 −5.6192 −0.5382568 ILE212 CB 1.419 −6.4711 0.8968 2569 ILE212 CG1 −0.0541 −6.54061.3449 2570 ILE212 CG2 2.2939 −7.3081 1.8521 2571 ILE212 CD1 −0.5911−7.9762 1.1884 2572 ASN213 N 4.2062 −4.2553 1.1941 2573 ASN213 CA 5.6213−4.2737 0.8628 2574 ASN213 C 6.1141 −5.6779 1.0676 2575 ASN213 O 6.4063−6.0634 2.1877 2576 ASN213 CB 6.382 −3.3112 1.7973 2577 ASN213 CG 6.0491−1.8733 1.5174 2578 ASN213 OD1 4.928 −1.5529 1.1578 2579 ASN213 ND27.0439 −0.9828 1.6817 2580 LEU214 N 6.1863 −6.4488 −0.0348 2581 LEU214CA 6.5039 −7.8588 0.1037 2582 LEU214 C 7.8453 −8.0892 0.7358 2583 LEU214O 8.0269 −9.1297 1.3458 2584 LEU214 CB 6.4267 −8.5505 −1.2667 2585LEU214 CG 5.1616 −9.4224 −1.339 2586 LEU214 CD1 3.9038 −8.532 −1.34052587 LEU214 CD2 5.2104 −10.2479 −2.6365 2588 ALA215 N 8.7748 −7.11820.6196 2589 ALA215 CA 10.0464 −7.2635 1.3105 2590 ALA215 C 9.7769−7.5799 2.7559 2591 ALA215 O 10.3279 −8.5342 3.2802 2592 ALA215 CB10.8273 −5.9415 1.1907 2593 GLU216 N 8.8848 −6.7868 3.3804 2594 GLU216CA 8.3903 −7.1675 4.6907 2595 GLU216 C 6.9476 −7.5615 4.5145 2596 GLU216O 6.0518 −6.9214 5.0423 2597 GLU216 CB 8.5538 −5.9747 5.6525 2598 GLU216CG 8.2854 −6.4355 7.0971 2599 GLU216 CD 7.3825 −5.4464 7.7742 2600GLU216 OE1 6.1396 −5.5673 7.6064 2601 GLU216 OE2 7.9134 −4.5541 8.48782602 ALA217 N 6.7323 −8.6343 3.7284 2603 ALA217 CA 5.374 −8.9999 3.36182604 ALA217 C 4.6093 −9.6378 4.4887 2605 ALA217 O 5.1317 −9.8014 5.57982606 ALA217 CB 5.4467 −9.9884 2.1838 2607 HIS218 N 3.3448 −10.00494.1972 2608 HIS218 CA 2.5306 −10.6643 5.2054 2609 HIS218 C 3.0285−12.0533 5.4931 2610 HIS218 O 3.9116 −12.551 4.8126 2611 HIS218 CB1.1008 −10.8309 4.6532 2612 HIS218 CG 0.3339 −9.543 4.6987 2613 HIS218ND1 0.8034 −8.4292 4.186 2614 HIS218 CD2 −0.8854 −9.3962 5.2515 2615HIS218 CE1 −0.0876 −7.51 4.3834 2616 HIS218 NE2 −1.0761 −7.9962 4.98562617 GLY219 N 2.427 −12.6904 6.5166 2618 GLY219 CA 2.6988 −14.10146.7225 2619 GLY219 C 1.8493 −14.8442 5.733 2620 GLY219 O 2.3558 −15.64984.9688 2621 GLN238 N 8.1324 −5.9044 −5.4568 2622 GLN238 CA 9.4731−5.6833 −4.9464 2623 GLN238 C 10.3999 −5.3856 −6.106 2624 GLN238 O9.9466 −5.2424 −7.2302 2625 GLN238 CB 9.5334 −4.5688 −3.8746 2626 GLN238CG 9.065 −5.0445 −2.4777 2627 GLN238 CD 8.29 −6.3276 −2.4983 2628 GLN238OE1 8.8192 −7.361 −2.1257 2629 GLN238 NE2 7.0217 −6.2661 −2.9362 2630THR239 N 11.721 −5.3061 −5.8507 2631 THR239 CA 12.6638 −5.2142 −6.95472632 THR239 C 13.5003 −6.4648 −6.9995 2633 THR239 O 14.0679 −6.8369−5.9846 2634 THR239 CB 13.5534 −3.9622 −6.8255 2635 THR239 OG1 14.3086−4.0071 −5.6115 2636 THR239 CG2 12.6716 −2.7002 −6.8315 2637 HIS240 N13.555 −7.1173 −8.1813 2638 HIS240 CA 14.163 −8.4408 −8.284 2639 HIS240C 15.4515 −8.5949 −7.5159 2640 HIS240 O 16.24 −7.6636 −7.4811 2641HIS240 CB 13.1137 −9.4858 −7.8556 2642 HIS240 CG 12.9509 −10.5509−8.9012 2643 HIS240 ND1 12.9088 −10.2764 −10.1849 2644 HIS240 CD212.8277 −11.8668 −8.6425 2645 HIS240 CE1 12.7565 −11.3982 −10.8156 2646HIS240 NE2 12.7006 −12.3442 −9.9931 2647 SER241 N 15.6651 −9.7692−6.8839 2648 SER241 CA 16.8516 −9.9218 −6.0565 2649 SER241 C 17.0666−11.3541 −5.6464 2650 SER241 O 16.3248 −12.2304 −6.0604 2651 SER241 CB16.6801 −9.0557 −4.7945 2652 SER241 OG 17.9373 −8.8969 −4.1322 2653GLY242 N 18.1042 −11.5854 −4.8158 2654 GLY242 CA 18.3714 −12.9407−4.3647 2655 GLY242 C 17.6696 −13.2358 −3.0682 2656 GLY242 O 16.7701−12.5049 −2.686 2657 LYS243 N 18.1019 −14.327 −2.4015 2658 LYS243 CA17.4273 −14.7712 −1.1904 2659 LYS243 C 17.4213 −13.7439 −0.0914 2660LYS243 O 18.3491 −12.9592 0.0143 2661 LYS243 CB 18.0192 −16.1181 −0.72842662 LYS243 CG 17.3879 −16.5818 0.5981 2663 LYS243 CD 18.3077 −16.16741.7608 2664 LYS243 CE 17.5847 −16.3813 3.1027 2665 LYS243 NZ 18.4874−16.0221 4.2054 2666 ILE244 N 16.3402 −13.7602 0.7163 2667 ILE244 CA16.1453 −12.7149 1.7103 2668 ILE244 C 17.1361 −12.8139 2.8396 2669ILE244 O 18.1547 −13.4736 2.7099 2670 ILE244 CB 14.7014 −12.7747 2.25752671 ILE244 CG1 13.6943 −13.0402 1.1243 2672 ILE244 CG2 14.3331 −11.43832.9311 2673 ILE244 CD1 12.3433 −13.4901 1.7111 2674 LEU245 N 16.8305−12.1362 3.9658 2675 LEU245 CA 17.7767 −12.0954 5.0684 2676 LEU245 C19.0752 −11.5053 4.5937 2677 LEU245 O 20.1429 −11.8815 5.0504 2678LEU245 CB 17.9564 −13.4847 5.7113 2679 LEU245 CG 17.7838 −13.383 7.23842680 LEU245 CD1 17.8183 −14.7962 7.8482 2681 LEU245 CD2 18.9207 −12.53777.8437 2682 GLU246 N 18.9479 −10.5707 3.6328 2683 GLU246 CA 20.1286−10.0014 3.0152 2684 GLU246 C 19.7591 −8.6028 2.6115 2685 GLU246 O19.5634 −8.3168 1.4408 2686 GLU246 CB 20.5101 −10.8743 1.8014 2687GLU246 CG 21.0342 −12.237 2.2939 2688 GLU246 CD 21.314 −13.1542 1.13972689 GLU246 OE1 20.5724 −14.1619 0.9912 2690 GLU246 OE2 22.2844 −12.87730.3853 2691 ASN247 N 19.6554 −7.7232 3.6269 2692 ASN247 CA 19.1986−6.3703 3.359 2693 ASN247 C 20.1145 −5.6244 2.4312 2694 ASN247 O 21.2558−6.0137 2.2406 2695 ASN247 CB 19.0449 −5.5882 4.6782 2696 ASN247 CG17.9811 −6.1721 5.5668 2697 ASN247 OD1 17.3251 −7.1387 5.2131 2698ASN247 ND2 17.8028 −5.571 6.7565 2699 GLY248 N 19.5829 −4.5368 1.84242700 GLY248 CA 20.3934 −3.7772 0.9097 2701 GLY248 C 19.509 −3.0448−0.0569 2702 GLY248 O 18.429 −3.5146 −0.3793 2703 LEU249 N 19.9903−1.8762 −0.523 2704 LEU249 CA 19.2109 −1.1168 −1.4862 2705 LEU249 C19.2242 −1.7899 −2.8314 2706 LEU249 O 19.6245 −2.9387 −2.9378 2707LEU249 CB 19.8299 0.2894 −1.6043 2708 LEU249 CG 19.594 1.0806 −0.30352709 LEU249 CD1 20.3173 2.4368 −0.3936 2710 LEU249 CD2 18.0856 1.3192−0.1002 2711 ARG261 N 3.7075 −19.562 −7.3982 2712 ARG261 CA 3.3851−20.9648 −7.232 2713 ARG261 C 4.5578 −21.6483 −6.5934 2714 ARG261 O5.6844 −21.4482 −7.02 2715 ARG261 CB 3.0626 −21.6256 −8.5898 2716 ARG261CG 4.1152 −21.2544 −9.6556 2717 ARG261 CD 3.7549 −21.9296 −10.9934 2718ARG261 NE 4.2205 −21.1202 −12.1063 2719 ARG261 CZ 4.8263 −21.6535−13.1285 2720 ARG261 NH1 5.223 −20.8915 −14.1043 2721 ARG261 NH2 5.0417−22.9348 −13.1953 2722 LYS262 N 4.2857 −22.4607 −5.5544 2723 LYS262 CA5.3856 −23.1774 −4.9376 2724 LYS262 C 5.8255 −24.3186 −5.8107 2725LYS262 O 5.1828 −24.6312 −6.8005 2726 LYS262 CB 5.0152 −23.6907 −3.53362727 LYS262 CG 6.3179 −23.9676 −2.7607 2728 LYS262 CD 6.0206 −24.5326−1.3622 2729 LYS262 CE 7.3098 −24.4656 −0.5237 2730 LYS262 NZ 7.0732−25.0873 0.7867 2731 VAL263 N 6.9519 −24.9407 −5.421 2732 VAL263 CA7.4726 −26.0424 −6.208 2733 VAL263 C 8.1395 −26.9928 −5.254 2734 VAL263O 7.6714 −27.148 −4.138 2735 VAL263 CB 8.4965 −25.4823 −7.2192 2736VAL263 CG1 7.793 −24.6213 −8.2849 2737 VAL263 CG2 9.5603 −24.642 −6.4892738 THR264 N 9.2483 −27.6135 −5.6989 2739 THR264 CA 10.0418 −28.4341−4.8006 2740 THR264 C 11.2795 −28.8729 −5.5293 2741 THR264 O 11.3053−28.8214 −6.7478 2742 THR264 CB 9.2501 −29.5985 −4.168 2743 THR264 OG110.1114 −30.4496 −3.4071 2744 THR264 CG2 8.5052 −30.4127 −5.2411 2745CYS265 N 12.3218 −29.2873 −4.785 2746 CYS265 CA 13.5704 −29.6047 −5.45282747 CYS265 C 14.2751 −30.6831 −4.6861 2748 CYS265 O 15.0484 −30.3903−3.7888 2749 CYS265 CB 14.4239 −28.3244 −5.5279 2750 CYS265 SG 14.0005−27.4808 −7.0785 2751 LYS266 N 13.9958 −31.9498 −5.0519 2752 LYS266 CA14.5391 −33.0536 −4.2768 2753 LYS266 C 13.9713 −32.9782 −2.887 2754LYS266 O 14.7014 −32.9772 −1.9084 2755 LYS266 CB 16.0807 −33.0492−4.2688 2756 LYS266 CG 16.6181 −33.1531 −5.7063 2757 LYS266 CD 18.0988−32.7333 −5.7235 2758 LYS266 CE 18.6018 −32.6909 −7.1778 2759 LYS266 NZ19.5072 −31.5454 −7.3469 2760 HIS267 N 12.6274 −32.897 −2.8274 2761HIS267 CA 11.9625 −32.7559 −1.5442 2762 HIS267 C 12.337 −31.4426 −0.91652763 HIS267 O 12.6154 −31.3721 0.2699 2764 HIS267 CB 12.2143 −33.9712−0.6284 2765 HIS267 CG 11.8724 −35.2288 −1.3735 2766 HIS267 ND1 12.6398−35.6975 −2.3307 2767 HIS267 CD2 10.7805 −35.9862 −1.1556 2768 HIS267CE1 12.0809 −36.7767 −2.7783 2769 HIS267 NE2 11.0167 −36.9974 −2.15012770 ARG268 N 12.3454 −30.3879 −1.7539 2771 ARG268 CA 12.6959 −29.0768−1.2425 2772 ARG268 C 11.7987 −28.073 −1.9135 2773 ARG268 O 12.2499−27.365 −2.7992 2774 ARG268 CB 14.1807 −28.7867 −1.539 2775 ARG268 CG15.075 −29.8563 −0.8856 2776 ARG268 CD 16.5401 −29.5911 −1.2753 2777ARG268 NE 17.2372 −30.8544 −1.429 2778 ARG268 CZ 17.8507 −31.1554−2.5371 2779 ARG268 NH1 18.4688 −32.2949 −2.6312 2780 ARG268 NH2 17.8589−30.3395 −3.5505 2781 PRO269 N 10.5143 −27.9991 −1.5013 2782 PRO269 CA9.5866 −27.0874 −2.1375 2783 PRO269 C 10.0311 −25.6531 −2.0958 2784PRO269 O 10.8327 −25.2856 −1.2517 2785 PRO269 CB 8.2973 −27.2861 −1.31842786 PRO269 CG 8.542 −28.4617 −0.3464 2787 PRO269 CD 10.0307 −28.8527−0.437 2788 GLU270 N 9.5086 −24.8419 −3.0367 2789 GLU270 CA 9.9543−23.4606 −3.0969 2790 GLU270 C 8.8187 −22.5727 −3.5148 2791 GLU270 O8.3378 −22.6901 −4.6302 2792 GLU270 CB 11.1188 −23.3223 −4.0978 2793GLU270 CG 12.3248 −24.1506 −3.6156 2794 GLU270 CD 12.7022 −25.1816−4.6395 2795 GLU270 OE1 13.9116 −25.2456 −4.9833 2796 GLU270 OE2 11.7987−25.9258 −5.1071 2797 TYR271 N 8.3949 −21.6792 −2.5981 2798 TYR271 CA7.3008 −20.7762 −2.9181 2799 TYR271 C 7.7473 −19.7883 −3.9578 2800TYR271 O 8.189 −18.6948 −3.6512 2801 TYR271 CB 6.8694 −20.0676 −1.62132802 TYR271 CG 5.9845 −21.0113 −0.8158 2803 TYR271 CD1 6.4001 −21.45330.4424 2804 TYR271 CD2 4.7584 −21.4337 −1.3361 2805 TYR271 CE1 5.5858−22.3144 1.1823 2806 TYR271 CE2 3.9429 −22.2927 −0.5958 2807 TYR271 CZ4.3602 −22.7375 0.6613 2808 TYR271 OH 3.5561 −23.6022 1.3935 2809 SER289N 17.1658 −18.93 −8.2538 2810 SER289 CA 18.3422 −19.0254 −7.408 2811SER289 C 18.4464 −20.4103 −6.8327 2812 SER289 O 17.4627 −21.1337 −6.81672813 SER289 CB 18.2595 −17.9919 −6.2689 2814 SER289 OG 18.1295 −16.678−6.8191 2815 ASP290 N 19.658 −20.783 −6.3711 2816 ASP290 CA 19.8478−22.1303 −5.8561 2817 ASP290 C 19.5131 −23.1241 −6.936 2818 ASP290 O18.616 −23.9338 −6.7705 2819 ASP290 CB 19.0064 −22.3277 −4.577 2820ASP290 CG 19.3558 −23.6273 −3.9113 2821 ASP290 OD1 18.5143 −24.5634−3.9658 2822 ASP290 OD2 20.4676 −23.7127 −3.3249 2823 SER291 N 20.2476−23.0372 −8.0632 2824 SER291 CA 19.905 −23.8381 −9.229 2825 SER291 C18.8119 −23.1601 −10.0025 2826 SER291 O 17.6878 −23.0693 −9.5349 2827SER291 CB 19.5625 −25.3123 −8.9314 2828 SER291 OG 20.6095 −25.9035−8.1578 2829 SER292 N 19.1722 −22.6698 −11.2039 2830 SER292 CA 18.195−21.9554 −12.0051 2831 SER292 C 17.2106 −22.9225 −12.5874 2832 SER292 O16.0318 −22.843 −12.2828 2833 SER292 CB 18.9224 −21.2063 −13.1356 2834SER292 OG 19.8986 −20.3309 −12.5658 2835 SER293 N 17.7122 −23.8448−13.4301 2836 SER293 CA 16.8111 −24.8207 −14.0092 2837 SER293 C 16.5572−25.9456 −13.0439 2838 SER293 O 16.5136 −27.0961 −13.4508 2839 SER293 CB17.3567 −25.3185 −15.3625 2840 SER293 OG 18.6298 −25.9437 −15.1757 2841LYS294 N 16.3779 −25.616 −11.7479 2842 LYS294 CA 16.0064 −26.6686−10.8191 2843 LYS294 C 14.5559 −27.0301 −10.9887 2844 LYS294 O 13.9326−27.5604 −10.0834 2845 LYS294 CB 16.4338 −26.4047 −9.3613 2846 LYS294 CG15.808 −25.1066 −8.8236 2847 LYS294 CD 16.0614 −25.0134 −7.3086 2848LYS294 CE 15.7922 −23.576 −6.8301 2849 LYS294 NZ 16.1052 −23.4562−5.3993 2850 TRP295 N 14.0282 −26.7341 −12.1931 2851 TRP295 CA 12.6821−27.1618 −12.5219 2852 TRP295 C 12.7817 −28.5945 −12.97 2853 TRP295 O12.2805 −28.9619 −14.0205 2854 TRP295 CB 12.1207 −26.2722 −13.6524 2855TRP295 CG 12.5239 −24.8384 −13.4586 2856 TRP295 CD1 13.5624 −24.2088−14.0301 2857 TRP295 CD2 11.8326 −23.8389 −12.5722 2858 TRP295 NE113.6136 −22.9662 −13.6174 2859 TRP295 CE2 12.6152 −22.7131 −12.7465 2860TRP295 CE3 10.7148 −23.8688 −11.7405 2861 TRP295 CZ2 12.3445 −21.5215−12.0738 2862 TRP295 CZ3 10.4389 −22.6751 −11.0637 2863 TRP295 CH211.2391 −21.5329 −11.2145 2864 GLU296 N 13.4637 −29.41 −12.1425 2865GLU296 CA 13.6738 −30.7962 −12.5123 2866 GLU296 C 13.6457 −31.6147−11.2556 2867 GLU296 O 12.8159 −32.5018 −11.1372 2868 GLU296 CB 15.0491−30.9523 −13.1904 2869 GLU296 CG 15.1797 −29.9717 −14.3715 2870 GLU296CD 16.5672 −30.0476 −14.9383 2871 GLU296 OE1 16.7082 −30.5508 −16.08452872 GLU296 OE2 17.5189 −29.5994 −14.2445 2873 LEU297 N 14.5557 −31.3013−10.3102 2874 LEU297 CA 14.5168 −31.9927 −9.0331 2875 LEU297 C 13.2502−31.6135 −8.3246 2876 LEU297 O 13.0172 −30.4371 −8.0988 2877 LEU297 CB15.7497 −31.6707 −8.1644 2878 LEU297 CG 15.873 −30.1595 −7.8868 2879LEU297 CD1 16.8503 −29.9272 −6.7201 2880 LEU297 CD2 16.3974 −29.4396−9.1426 2881 LEU298 N 12.4169 −32.625 −8.0092 2882 LEU298 CA 11.1004−32.3273 −7.4683 2883 LEU298 C 10.3621 −31.3525 −8.3461 2884 LEU298 O9.5067 −30.6151 −7.8844 2885 LEU298 CB 11.1792 −31.8834 −5.9944 2886LEU298 CG 10.8252 −33.0547 −5.057 2887 LEU298 CD1 9.3623 −33.4862−5.2695 2888 LEU298 CD2 11.7632 −34.252 −5.3043 2889 SER299 N 10.7258−31.3566 −9.6423 2890 SER299 CA 10.0974 −30.4369 −10.5692 2891 SER299 C9.9685 −31.1633 −11.8808 2892 SER299 O 10.6607 −30.836 −12.8313 2893SER299 CB 10.9557 −29.1635 −10.7041 2894 SER299 OG 11.1545 −28.5444−9.4319 2895 LEU307 N 6.3956 −23.5665 −16.4828 2896 LEU307 CA 7.1931−22.4652 −15.9735 2897 LEU307 C 8.1083 −21.8538 −16.9929 2898 LEU307 O8.3851 −22.46 −18.0157 2899 LEU307 CB 7.9177 −22.8665 −14.6752 2900LEU307 CG 8.3099 −21.5991 −13.8939 2901 LEU307 CD1 7.8988 −21.7401−12.417 2902 LEU307 CD2 9.832 −21.3982 −13.9943 2903 ARG308 N 8.5562−20.6166 −16.6943 2904 ARG308 CA 9.3351 −19.8688 −17.6684 2905 ARG308 C8.5562 −19.7259 −18.9476 2906 ARG308 O 9.1174 −19.6909 −20.0309 2907ARG308 CB 10.7482 −20.4495 −17.8796 2908 ARG308 CG 11.4783 −20.6338−16.5325 2909 ARG308 CD 11.4961 −19.3378 −15.6937 2910 ARG308 NE 11.6184−18.1599 −16.534 2911 ARG308 CZ 12.7781 −17.7454 −16.9542 2912 ARG308NH1 13.8705 −18.3784 −16.6421 2913 ARG308 NH2 12.8453 −16.6798 −17.69532914 LYS309 N 7.2213 −19.6664 −18.7836 2915 LYS309 CA 6.3417 −19.6382−19.9366 2916 LYS309 C 6.2972 −18.2635 −20.5374 2917 LYS309 O 6.5442−18.1125 −21.723 2918 LYS309 CB 4.9278 −20.0332 −19.4624 2919 LYS309 CG4.57 −19.2689 −18.1717 2920 LYS309 CD 3.2022 −19.7298 −17.6396 2921LYS309 CE 3.0136 −19.1806 −16.2137 2922 LYS309 NZ 1.7878 −19.7429−15.6295 2923 ASP310 N 5.9687 −17.2608 −19.7008 2924 ASP310 CA 5.7973−15.9166 −20.2246 2925 ASP310 C 7.1124 −15.3194 −20.6424 2926 ASP310 O8.1451 −15.9616 −20.5346 2927 ASP310 CB 5.1166 −15.0146 −19.1735 2928ASP310 CG 4.1993 −15.8056 −18.2838 2929 ASP310 OD1 4.64 −16.1806−17.1645 2930 ASP310 OD2 3.0342 −16.0465 −18.6978 2931 ASN311 N 7.063−14.0653 −21.1296 2932 ASN311 CA 8.2916 −13.436 −21.579 2933 ASN311 C8.2891 −11.9763 −21.2219 2934 ASN311 O 7.4456 −11.544 −20.4518 2935ASN311 CB 8.4266 −13.6396 −23.0999 2936 ASN311 CG 8.7356 −15.0815−23.3811 2937 ASN311 OD1 7.923 −15.7738 −23.9713 2938 ASN311 ND2 9.9227−15.549 −22.9547 2939 ASP312 N 9.2508 −11.2127 −21.7825 2940 ASP312 CA9.3773 −9.8163 −21.3949 2941 ASP312 C 9.6773 −9.7719 −19.9227 2942ASP312 O 9.0169 −9.0665 −19.1788 2943 ASP312 CB 8.0958 −9.0254 −21.73482944 ASP312 CG 8.4216 −7.784 −22.5124 2945 ASP312 OD1 8.8258 −6.7738−21.8768 2946 ASP312 OD2 8.2557 −7.8116 −23.7612 2947 GLY313 N 10.6782−10.5659 −19.4978 2948 GLY313 CA 10.9377 −10.6612 −18.074 2949 GLY313 C10.2069 −11.8465 −17.5087 2950 GLY313 O 9.4537 −11.6938 −16.5604 2951SER340 N −16.3887 −9.2646 −18.8495 2952 SER340 CA −16.9789 −10.5545−18.5298 2953 SER340 C −16.0382 −11.6865 −18.8295 2954 SER340 O −16.4224−12.6865 −19.4149 2955 SER340 CB −18.4188 −10.7336 −19.0575 2956 SER340OG −18.4344 −10.989 −20.4645 2957 GLN341 N −14.7733 −11.4999 −18.4092958 GLN341 CA −13.7872 −12.5328 −18.6593 2959 GLN341 C −13.9973−13.6595 −17.6917 2960 GLN341 O −13.8658 −13.4705 −16.4927 2961 GLN341CB −12.3744 −11.9426 −18.5055 2962 GLN341 CG −12.2671 −10.6679 −19.36352963 GLN341 CD −10.834 −10.3784 −19.6945 2964 GLN341 OE1 −10.5296−10.0819 −20.8379 2965 GLN341 NE2 −9.9401 −10.467 −18.693 2966 GLU342 N−14.334 −14.842 −18.2416 2967 GLU342 CA −14.5771 −15.9907 −17.3859 2968GLU342 C −13.3583 −16.3002 −16.5657 2969 GLU342 O −13.4924 −16.6687−15.4098 2970 GLU342 CB −14.8923 −17.2033 −18.2805 2971 GLU342 CG−16.2407 −16.984 −18.991 2972 GLU342 CD −16.5529 −18.1797 −19.8423 2973GLU342 OE1 −17.2257 −19.1124 −19.3278 2974 GLU342 OE2 −16.1387 −18.1828−21.0323 2975 ALA343 N −12.1655 −16.1326 −17.171 2976 ALA343 CA −10.9489−16.4186 −16.4325 2977 ALA343 C −10.8286 −15.5741 −15.1958 2978 ALA343 O−11.2757 −14.4382 −15.1781 2979 ALA343 CB −9.7128 −16.2208 −17.3284 2980ALA344 N −10.219 −16.1722 −14.1534 2981 ALA344 CA −10.0489 −15.4662−12.8946 2982 ALA344 C −11.3061 −15.3614 −12.0785 2983 ALA344 O −12.3777−15.1106 −12.6066 2984 ALA344 CB −9.3563 −14.097 −13.0367 2985 GLN345 N−11.1378 −15.5569 −10.756 2986 GLN345 CA −12.2396 −15.3412 −9.8354 2987GLN345 C −12.542 −13.8713 −9.7507 2988 GLN345 O −11.7928 −13.0625−10.2761 2989 GLN345 CB −11.831 −15.8247 −8.4297 2990 GLN345 CG −11.2853−17.2639 −8.4949 2991 GLN345 CD −11.6086 −17.9922 −7.2226 2992 GLN345OE1 −12.3591 −18.9535 −7.2488 2993 GLN345 NE2 −11.0403 −17.5418 −6.08992994 MET351 N −19.5867 0.1074 −2.8647 2995 MET351 CA −18.9917 1.333−2.3614 2996 MET351 C −19.9682 2.156 −1.5709 2997 MET351 O −21.17052.0284 −1.7395 2998 MET351 CB −18.407 2.171 −3.5126 2999 MET351 CG−17.4789 3.2657 −2.9539 3000 MET351 SD −17.0298 4.3543 −4.336 3001MET351 CE −16.091 5.592 −3.3933 3002 ARG352 N −19.4169 3.0104 −0.69 3003ARG352 CA −20.2817 3.8805 0.083 3004 ARG352 C −19.7783 5.2922 −0.00363005 ARG352 O −18.6784 5.5259 −0.4795 3006 ARG352 CB −20.3127 3.43211.5564 3007 ARG352 CG −21.0796 2.1031 1.6878 3008 ARG352 CD −21.1451.7058 3.174 3009 ARG352 NE −22.4189 1.0678 3.4523 3010 ARG352 CZ−22.4939 0.0081 4.2047 3011 ARG352 NH1 −23.6545 −0.5309 4.4316 3012ARG352 NH2 −21.4306 −0.5187 4.7369 3013 GLU353 N −20.6069 6.243 0.46563014 GLU353 CA −20.1771 7.6289 0.4297 3015 GLU353 C −20.6351 8.34011.6712 3016 GLU353 O −21.3374 7.7625 2.4861 3017 GLU353 CB −20.72138.3284 −0.8312 3018 GLU353 CG −20.068 7.729 −2.0919 3019 GLU353 CD−18.7307 8.3669 −2.3365 3020 GLU353 OE1 −17.7195 7.8583 −1.7831 3021GLU353 OE2 −18.6851 9.3703 −3.097 3022 SER58 N 4.5161 25.4695 −12.04483023 SER58 CA 5.8856 25.2609 −11.6081 3024 SER58 C 6.2426 26.247−10.5313 3025 SER58 O 5.5046 27.1917 −10.3003 3026 SER58 CB 6.866225.352 −12.7932 3027 SER58 OG 6.8328 26.6638 −13.3617 3028 GLY59 N7.3844 26.0176 −9.8559 3029 GLY59 CA 7.7292 26.9004 −8.758 3030 GLY59 C9.2172 27.0021 −8.6004 3031 GLY59 O 9.8707 26.0045 −8.3406 3032 SER60 N9.7354 28.2375 −8.7523 3033 SER60 CA 11.1556 28.4693 −8.5488 3034 SER60C 12.0012 27.647 −9.479 3035 SER60 O 12.1356 28.0169 −10.6331 3036 SER60CB 11.5573 28.3065 −7.0694 3037 SER60 OG 12.8944 28.7744 −6.8761 3038LEU61 N 12.5734 26.5358 −8.974 3039 LEU61 CA 13.3703 25.6829 −9.83883040 LEU61 C 12.4744 25.0345 −10.8529 3041 LEU61 O 12.8547 24.9202−12.0066 3042 LEU61 CB 14.0287 24.5946 −8.9719 3043 LEU61 CG 15.154325.2173 −8.1245 3044 LEU61 CD1 15.6818 24.1691 −7.1278 3045 LEU61 CD216.3056 25.6736 −9.0407 3046 LEU62 N 11.2662 24.634 −10.4083 3047 LEU62CA 10.2797 24.1384 −11.3521 3048 LEU62 C 9.9899 25.2189 −12.3535 3049LEU62 O 9.8158 24.9231 −13.5236 3050 LEU62 CB 8.9859 23.8371 −10.57383051 LEU62 CG 9.182 22.6054 −9.6716 3052 LEU62 CD1 8.0821 22.5794−8.5947 3053 LEU62 CD2 9.0985 21.3253 −10.5239 3054 GLN63 N 9.960926.4803 −11.8805 3055 GLN63 CA 9.7744 27.5884 −12.8005 3056 GLN63 C11.0014 27.8053 −13.6448 3057 GLN63 O 10.9047 28.4324 −14.6872 3058GLN63 CB 9.4807 28.8481 −11.9674 3059 GLN63 CG 8.0421 28.7587 −11.42553060 GLN63 CD 7.7327 29.9547 −10.5738 3061 GLN63 OE1 7.4182 29.8047−9.4048 3062 GLN63 NE2 7.817 31.1633 −11.1583 3063 LYS64 N 12.158827.2732 −13.205 3064 LYS64 CA 13.3485 27.3597 −14.0352 3065 LYS64 C13.3802 26.1327 −14.9053 3066 LYS64 O 14.3184 25.3531 −14.8528 3067LYS64 CB 14.5923 27.3962 −13.124 3068 LYS64 CG 14.4931 28.5556 −12.11683069 LYS64 CD 15.2394 28.156 −10.8313 3070 LYS64 CE 14.8604 29.1181−9.6912 3071 LYS64 NZ 15.2794 28.5352 −8.4089 3072 SER167 N 7.92086.4674 −28.337 3073 SER167 CA 7.7436 6.2762 −29.7669 3074 SER167 C7.2045 4.9046 −30.0648 3075 SER167 O 6.6653 4.6971 −31.1401 3076 SER167CB 9.1172 6.4355 −30.4468 3077 SER167 OG 10.0458 5.4869 −29.9125 3078THR168 N 7.3541 3.9725 −29.1012 3079 THR168 CA 6.9327 2.6017 −29.33993080 THR168 C 5.4961 2.4848 −29.7688 3081 THR168 O 4.7103 3.393 −29.55183082 THR168 CB 7.1788 1.756 −28.0715 3083 THR168 OG1 6.9197 0.3739−28.3344 3084 THR168 CG2 6.2787 2.2329 −26.9143 3085 TYR169 N 5.16491.3297 −30.3795 3086 TYR169 CA 3.7747 1.0608 −30.7045 3087 TYR169 C2.9715 1.1166 −29.4396 3088 TYR169 O 1.8501 1.5972 −29.4459 3089 TYR169CB 3.6924 −0.3845 −31.2285 3090 TYR169 CG 3.9609 −0.4244 −32.7279 3091TYR169 CD1 5.1791 0.031 −33.2386 3092 TYR169 CD2 2.9836 −0.9252 −33.59143093 TYR169 CE1 5.4261 −0.0331 −34.6122 3094 TYR169 CE2 3.2344 −0.9998−34.9634 3095 TYR169 CZ 4.4537 −0.5472 −35.4739 3096 TYR169 OH 4.6986−0.6064 −36.8403 3097 LYS170 N 3.5939 0.6215 −28.3524 3098 LYS170 CA2.9178 0.5885 −27.0715 3099 LYS170 C 2.4888 1.9565 −26.6329 3100 LYS170O 1.4293 2.063 −26.0395 3101 LYS170 CB 3.9232 0.0584 −26.0343 3102LYS170 CG 3.2029 −0.8809 −25.0516 3103 LYS170 CD 4.1528 −1.1913 −23.88213104 LYS170 CE 3.8263 −2.5692 −23.2794 3105 LYS170 NZ 4.6894 −2.8051−22.1133 3106 ASN171 N 3.2958 3.0014 −26.9052 3107 ASN171 CA 2.95314.2881 −26.3252 3108 ASN171 C 3.0516 5.4378 −27.2882 3109 ASN171 O 3.2756.5589 −26.8592 3110 ASN171 CB 3.7789 4.5344 −25.0468 3111 ASN171 CG3.7314 3.3219 −24.1631 3112 ASN171 OD1 4.7508 2.6909 −23.9381 3113ASN171 ND2 2.5327 2.9848 −23.6566 3114 LEU172 N 2.8874 5.1636 −28.59763115 LEU172 CA 3.054 6.2303 −29.5703 3116 LEU172 C 1.9448 7.2373−29.4526 3117 LEU172 O 0.8427 6.9818 −29.8769 3118 LEU172 CB 3.10055.6355 −30.9919 3119 LEU172 CG 3.9739 6.524 −31.8995 3120 LEU172 CD14.1502 5.8546 −33.2738 3121 LEU172 CD2 3.3112 7.8991 −32.0998 3122GLN367 N −11.6584 7.6284 20.3537 3123 GLN367 CA −11.2992 8.0993 21.6783124 GLN367 C −11.1646 9.5941 21.7165 3125 GLN367 O −10.6996 10.127722.7113 3126 GLN367 CB −9.9796 7.4354 22.1105 3127 GLN367 CG −10.02257.1421 23.6209 3128 GLN367 CD −9.0236 6.071 23.9479 3129 GLN367 OE1−9.4052 4.9967 24.3819 3130 GLN367 NE2 −7.7255 6.3572 23.7419 3131LEU368 N −11.5754 10.2737 20.6282 3132 LEU368 CA −11.5262 11.723120.6468 3133 LEU368 C −12.5936 12.2155 21.5826 3134 LEU368 O −13.745312.3421 21.199 3135 LEU368 CB −11.7396 12.2486 19.2157 3136 LEU368 CG−10.4387 12.1409 18.3976 3137 LEU368 CD1 −10.6866 12.677 16.9756 3138LEU368 CD2 −9.3312 12.9809 19.061 3139 LEU369 N −12.1779 12.482 22.83633140 LEU369 CA −13.138 12.8724 23.8552 3141 LEU369 C −13.8092 14.162323.484 3142 LEU369 O −15.0241 14.2483 23.5615 3143 LEU369 CB −12.402413.0369 25.1993 3144 LEU369 CG −11.5478 11.7894 25.5007 3145 LEU369 CD1−10.7853 11.9967 26.8221 3146 LEU369 CD2 −12.4446 10.5419 25.6142 3147GLN370 N −13.0054 15.1578 23.0591 3148 GLN370 CA −13.5889 16.394222.5661 3149 GLN370 C −14.5276 16.0684 21.4381 3150 GLN370 O −15.609916.6268 21.3618 3151 GLN370 CB −12.4205 17.2626 22.0543 3152 GLN370 CG−12.9177 18.3894 21.1269 3153 GLN370 CD −12.7668 17.9552 19.697 3154GLN370 OE1 −13.7564 17.7562 19.0116 3155 GLN370 NE2 −11.5136 17.804919.2321 3156 ASP371 N −14.091 15.14 20.567 3157 ASP371 CA −14.927 14.75519.446 3158 ASP371 C −16.1654 14.0217 19.8864 3159 ASP371 O −16.257213.6014 21.0288 3160 ASP371 CB −14.0746 13.845 18.5474 3161 ASP371 CG−13.1204 14.6693 17.7345 3162 ASP371 OD1 −13.3767 14.8343 16.5124 3163ASP371 OD2 −12.1057 15.1415 18.3128 3164 THR372 N −17.1371 13.879518.9632 3165 THR372 CA −18.3719 13.2085 19.3346 3166 THR372 C −18.284711.727 19.0963 3167 THR372 O −18.5994 10.9685 19.9991 3168 THR372 CB−19.5853 13.834 18.6163 3169 THR372 OG1 −20.7591 13.0569 18.8679 3170THR372 CG2 −19.3433 13.9323 17.0994 3171 SER382 N −21.7125 −7.23876.7774 3172 SER382 CA −21.778 −8.4416 7.5895 3173 SER382 C −21.0627−9.6106 6.9768 3174 SER382 O −20.601 −9.5345 5.8495 3175 SER382 CB−23.2573 −8.7962 7.832 3176 SER382 OG −23.8439 −7.8212 8.6971 3177VAL383 N −20.9902 −10.709 7.7553 3178 VAL383 CA −20.4217 −11.9392 7.23323179 VAL383 C −20.6916 −13.0827 8.177 3180 VAL383 O −20.5488 −12.92239.3776 3181 VAL383 CB −18.9717 −11.7586 6.7422 3182 VAL383 CG1 −18.0495−11.3373 7.8991 3183 VAL383 CG2 −18.4529 −13.0195 6.0279 3184 TRP384 N−21.1268 −14.2369 7.6338 3185 TRP384 CA −21.5699 −15.3172 8.4986 3186TRP384 C −20.5212 −16.3844 8.6219 3187 TRP384 O −19.4864 −16.2913 7.98533188 TRP384 CB −22.9502 −15.8666 8.0679 3189 TRP384 CG −22.8699 −16.94517.0232 3190 TRP384 CD1 −22.4297 −18.2021 7.1924 3191 TRP384 CD2 −23.2752−16.8131 5.5799 3192 TRP384 NE1 −22.4732 −18.8513 6.0555 3193 TRP384 CE2−22.9321 −18.0508 5.0717 3194 TRP384 CE3 −23.8559 −15.8137 4.8008 3195TRP384 CZ2 −23.0663 −18.3452 3.7152 3196 TRP384 CZ3 −23.9929 −16.13.4379 3197 TRP384 CH2 −23.5757 −17.3263 2.9019 3198 ARG385 N −20.7905−17.4056 9.4551 3199 ARG385 CA −19.783 −18.4237 9.6788 3200 ARG385 C−20.499 −19.7103 9.9672 3201 ARG385 O −20.8957 −19.9297 11.0998 3202ARG385 CB −19.0048 −17.9882 10.929 3203 ARG385 CG −17.802 −18.899911.2165 3204 ARG385 CD −17.0898 −18.3609 12.4697 3205 ARG385 NE −16.5758−17.0295 12.204 3206 ARG385 CZ −16.7127 −16.069 13.0714 3207 ARG385 NH1−16.2541 −14.8854 12.7933 3208 ARG385 NH2 −17.3001 −16.2711 14.2137 3209PRO386 N −20.6675 −20.5782 8.95 3210 PRO386 CA −21.3959 −21.8131 9.15583211 PRO386 C −20.581 −22.7837 9.9612 3212 PRO386 O −19.5745 −22.416210.546 3213 PRO386 CB −21.4659 −22.3502 7.7145 3214 PRO386 CG −20.3189−21.6558 6.9529 3215 PRO386 CD −20.1001 −20.302 7.6479 3216 GLU387 N−21.0369 −24.0503 9.9733 3217 GLU387 CA −20.2927 −25.0616 10.6974 3218GLU387 C −19.1297 −25.5457 9.8748 3219 GLU387 O −19.0221 −26.7281 9.59033220 GLU387 CB −21.2503 −26.2048 11.0833 3221 GLU387 CG −22.2475−25.6974 12.1415 3222 GLU387 CD −22.8692 −26.8624 12.8554 3223 GLU387OE1 −24.1183 −27.0029 12.7751 3224 GLU387 OE2 −22.1147 −27.6381 13.50143225 GLU388 N −18.2392 −24.6075 9.495 3226 GLU388 CA −17.0442 −25.00768.7712 3227 GLU388 C −16.0882 −25.7088 9.7019 3228 GLU388 O −16.4578−26.0365 10.8183 3229 GLU388 CB −16.4101 −23.7532 8.1366 3230 GLU388 CG−15.4454 −24.1537 7.0037 3231 GLU388 CD −14.086 −23.5654 7.2516 3232GLU388 OE1 −13.8775 −22.379 6.8856 3233 GLU388 OE2 −13.2139 −24.28947.8016 3234 GLY389 N −14.8465 −25.9491 9.2367 3235 GLY389 CA −13.9185−26.7395 10.0294 3236 GLY389 C −13.4262 −26.0583 11.2769 3237 GLY389 O−14.1027 −25.2136 11.8411 3238 ARG390 N −12.2154 −26.4654 11.7068 3239ARG390 CA −11.6679 −25.9396 12.9458 3240 ARG390 C −11.1701 −24.534912.7423 3241 ARG390 O −9.9756 −24.313 12.6198 3242 ARG390 CB −10.5192−26.8764 13.3736 3243 ARG390 CG −9.9781 −26.4675 14.7567 3244 ARG390 CD−10.9625 −26.9213 15.8493 3245 ARG390 NE −10.6131 −26.2863 17.1065 3246ARG390 CZ −11.4716 −25.5413 17.74 3247 ARG390 NH1 −11.1289 −24.988818.8657 3248 ARG390 NH2 −12.6657 −25.3433 17.2635 3249 ARG391 N −12.1066−23.5686 12.7144 3250 ARG391 CA −11.675 −22.2004 12.5066 3251 ARG391 C−11.4618 −21.4903 13.8149 3252 ARG391 O −11.8324 −20.3359 13.9572 3253ARG391 CB −12.6535 −21.4511 11.5852 3254 ARG391 CG −12.843 −22.218210.2661 3255 ARG391 CD −14.3048 −22.0698 9.8165 3256 ARG391 NE −15.1209−22.9598 10.6179 3257 ARG391 CZ −16.3317 −22.6372 10.9653 3258 ARG391NH1 −17.0163 −23.463 11.6969 3259 ARG391 NH2 −16.8663 −21.511 10.5973260 SER392 N −10.8434 −22.1905 14.7859 3261 SER392 CA −10.515 −21.519716.0313 3262 SER392 C −9.222 −20.7737 15.8405 3263 SER392 O −8.3233−20.8575 16.6623 3264 SER392 CB −10.4051 −22.5746 17.1487 3265 SER392 OG−10.2618 −21.9278 18.4149 3266 LEU393 N −9.1362 −20.0478 14.7083 3267LEU393 CA −7.9112 −19.3408 14.3803 3268 LEU393 C −8.1089 −18.717213.0275 3269 LEU393 O −7.5096 −19.1761 12.0691 3270 LEU393 CB −6.6802−20.2771 14.4205 3271 LEU393 CG −6.7754 −21.4227 13.3897 3272 LEU393 CD1−5.3984 −22.0945 13.2432 3273 LEU393 CD2 −7.8166 −22.4742 13.8166 3274ARG394 N −8.9651 −17.68 12.9082 3275 ARG394 CA −9.2458 −17.2221 11.55673276 ARG394 C −9.8339 −15.8452 11.3865 3277 ARG394 O −10.9908 −15.747311.0116 3278 ARG394 CB −10.0227 −18.2562 10.7129 3279 ARG394 CG −10.3857−19.5313 11.4973 3280 ARG394 CD −9.5337 −20.7126 10.9953 3281 ARG394 NE−9.8601 −20.9951 9.6108 3282 ARG394 CZ −10.0667 −22.2112 9.1969 3283ARG394 NH1 −9.9769 −23.2251 10.0066 3284 ARG394 NH2 −10.3721 −22.41427.9499 3285 PRO395 N −9.0548 −14.7611 11.5918 3286 PRO395 CA −9.4903−13.4464 11.1559 3287 PRO395 C −9.5917 −13.3885 9.6519 3288 PRO395 O−9.9065 −14.3791 9.0115 3289 PRO395 CB −8.2908 −12.5759 11.5797 3290PRO395 CG −7.1218 −13.5207 11.9251 3291 PRO395 CD −7.7492 −14.898812.1934 3292 CYS396 N −9.3171 −12.2055 9.0684 3293 CYS396 CA −9.4524−12.0876 7.6277 3294 CYS396 C −8.5442 −11.0416 7.0407 3295 CYS396 O−7.3374 −11.1843 7.1501 3296 CYS396 CB −10.9109 −11.954 7.1431 3297CYS396 SG −12.0989 −11.6904 8.4891 3298 SER397 N −9.1188 −10.002 6.39943299 SER397 CA −8.292 −9.0079 5.7316 3300 SER397 C −9.2078 −8.01295.0712 3301 SER397 O −9.3854 −8.057 3.8638 3302 SER397 CB −7.3971−9.7028 4.683 3303 SER397 OG −6.675 −8.7363 3.9151 3304 VAL398 N −9.8078−7.1174 5.8837 3305 VAL398 CA −10.8301 −6.221 5.3628 3306 VAL398 C−10.4519 −5.596 4.0479 3307 VAL398 O −9.4642 −4.8841 3.9578 3308 VAL398CB −11.2403 −5.1829 6.4317 3309 VAL398 CG1 −12.403 −5.6985 7.2958 3310VAL398 CG2 −11.6695 −3.8377 5.8196 3311 ARG413 N −3.5361 17.9222 −5.17793312 ARG413 CA −4.5546 17.0011 −5.6605 3313 ARG413 C −4.175 15.563−5.4325 3314 ARG413 O −4.4415 14.7133 −6.2676 3315 ARG413 CB −4.868417.2711 −7.1488 3316 ARG413 CG −4.846 18.782 −7.4541 3317 ARG413 CD−6.0673 19.4646 −6.8115 3318 ARG413 NE −6.5687 20.4804 −7.7188 3319ARG413 CZ −7.587 20.2434 −8.4945 3320 ARG413 NH1 −8.0068 21.1749 −9.2983321 ARG413 NH2 −8.1897 19.0908 −8.4829 3322 LYS414 N −3.5268 15.2994−4.2827 3323 LYS414 CA −3.097 13.9421 −4.006 3324 LYS414 C −4.256613.1746 −3.4316 3325 LYS414 O −4.6461 13.4592 −2.3106 3326 LYS414 CB−1.9473 14.0374 −2.985 3327 LYS414 CG −1.2538 12.6735 −2.821 3328 LYS414CD −0.0276 12.8477 −1.9063 3329 LYS414 CE 0.6361 11.4828 −1.6484 3330LYS414 NZ 1.2619 10.9927 −2.8844 3331 PRO415 N −4.8245 12.1984 −4.17293332 PRO415 CA −5.9278 11.4283 −3.6376 3333 PRO415 C −5.4305 10.5023−2.5651 3334 PRO415 O −4.2473 10.2053 −2.5311 3335 PRO415 CB −6.369710.5929 −4.8548 3336 PRO415 CG −5.305 10.7827 −5.9589 3337 PRO415 CD−4.336 11.8912 −5.4999 3338 LEU416 N −6.342 10.0587 −1.676 3339 LEU416CA −5.9331 9.1489 −0.6153 3340 LEU416 C −7.0039 9.0322 0.4323 3341LEU416 O −8.0493 9.6537 0.3288 3342 LEU416 CB −4.619 9.587 0.0726 3343LEU416 CG −4.8195 10.827 0.9679 3344 LEU416 CD1 −3.5219 11.1125 1.74523345 LEU416 CD2 −5.1887 12.0586 0.1214 3346 LEU417 N −6.7043 8.23121.4723 3347 LEU417 CA −7.5794 8.2124 2.6286 3348 LEU417 C −7.3649 9.47433.4198 3349 LEU417 O −6.304 10.0736 3.3467 3350 LEU417 CB −7.2075 6.98393.4839 3351 LEU417 CG −7.7439 7.1108 4.9227 3352 LEU417 CD1 −9.27496.9509 4.9301 3353 LEU417 CD2 −7.0885 6.0397 5.8121 3354 ALA418 N−8.3933 9.879 4.1866 3355 ALA418 CA −8.2268 11.0604 5.0123 3356 ALA418 C−7.9996 10.6567 6.4381 3357 ALA418 O −7.305 11.3518 7.1618 3358 ALA418CB −9.5567 11.8355 5.0052 3359 ARG425 N −18.6675 −5.1801 14.9036 3360ARG425 CA −19.6778 −6.0328 14.305 3361 ARG425 C −20.1478 −7.0639 15.29123362 ARG425 O −19.5831 −7.1842 16.3665 3363 ARG425 CB −19.0115 −6.806113.1523 3364 ARG425 CG −18.7297 −5.8777 11.9577 3365 ARG425 CD −17.818−6.6277 10.9718 3366 ARG425 NE −18.051 −6.2043 9.6033 3367 ARG425 CZ−18.372 −7.0629 8.6781 3368 ARG425 NH1 −18.5431 −6.6498 7.4579 3369ARG425 NH2 −18.5244 −8.3268 8.944 3370 MET426 N −21.1986 −7.8127 14.89833371 MET426 CA −21.719 −8.856 15.767 3372 MET426 C −22.8919 −9.49915.0809 3373 MET426 O −23.8577 −8.8107 14.7916 3374 MET426 CB −22.1868−8.2571 17.1093 3375 MET426 CG −21.173 −8.61 18.2132 3376 MET426 SD−21.2964 −7.3602 19.5248 3377 MET426 CE −20.4539 −8.2886 20.841 3378ASN427 N −22.806 −10.8212 14.8188 3379 ASN427 CA −23.905 −11.506 14.14923380 ASN427 C −24.0585 −11.0112 12.7365 3381 ASN427 O −24.7822 −10.055212.5058 3382 ASN427 CB −25.2258 −11.3252 14.9271 3383 ASN427 CG −25.2255−12.1399 16.1864 3384 ASN427 OD1 −25.405 −11.5895 17.2602 3385 ASN427ND2 −25.0267 −13.4644 16.0609 3386 LYS428 N −23.3755 −11.6623 11.77363387 LYS428 CA −23.4068 −11.1175 10.4257 3388 LYS428 C −23.5166 −12.17369.3602 3389 LYS428 O −23.4093 −13.3498 9.6638 3390 LYS428 CB −22.1673−10.2258 10.2332 3391 LYS428 CG −22.2938 −9.0068 11.1642 3392 LYS428 CD−21.2459 −7.9416 10.8006 3393 LYS428 CE −21.6234 −6.6238 11.5019 3394LYS428 NZ −22.8771 −6.1019 10.9393 3395 GLU429 N −23.745 −11.7541 8.09833396 GLU429 CA −23.9137 −12.747 7.0487 3397 GLU429 C −22.9051 −12.59245.9465 3398 GLU429 O −22.5972 −11.4855 5.5365 3399 GLU429 CB −25.3542−12.7924 6.5111 3400 GLU429 CG −26.2967 −13.2913 7.6237 3401 GLU429 CD−26.9033 −14.6167 7.2596 3402 GLU429 OE1 −27.0373 −15.4729 8.1741 3403GLU429 OE2 −27.2415 −14.815 6.0614 3404 MET430 N −22.3773 −13.75165.5091 3405 MET430 CA −21.1515 −13.7997 4.7279 3406 MET430 C −21.1395−12.9922 3.4604 3407 MET430 O −22.144 −12.4203 3.0689 3408 MET430 CB−20.8649 −15.2791 4.4098 3409 MET430 CG −19.7321 −15.8442 5.2888 3410MET430 SD −19.858 −17.6607 5.2326 3411 MET430 CE −18.4284 −18.14056.2421 3412 THR431 N −19.9535 −12.9534 2.8209 3413 THR431 CA −19.8141−12.1439 1.6235 3414 THR431 C −18.9606 −12.8425 0.6023 3415 THR431 O−19.3568 −12.9109 −0.5498 3416 THR431 CB −19.1786 −10.7891 1.993 3417THR431 OG1 −17.9959 −10.9957 2.7711 3418 THR431 CG2 −20.1824 −9.94462.7992 3419 TRP432 N −17.7884 −13.3659 1.0154 3420 TRP432 CA −16.9162−13.9906 0.0315 3421 TRP432 C −16.2157 −15.1928 0.5972 3422 TRP432 O−16.0256 −15.2741 1.7998 3423 TRP432 CB −15.8778 −13.0153 −0.563 3424TRP432 CG −16.01 −11.6374 0.0191 3425 TRP432 CD1 −15.3432 −11.1416 1.0723426 TRP432 CD2 −16.9314 −10.5544 −0.4726 3427 TRP432 NE1 −15.7193−9.9077 1.3003 3428 TRP432 CE2 −16.6595 −9.5272 0.4111 3429 TRP432 CE3−17.862 −10.4442 −1.5046 3430 TRP432 CZ2 −17.3132 −8.298 0.3284 3431TRP432 CZ3 −18.5277 −9.2155 −1.589 3432 TRP432 CH2 −18.2584 −8.1672−0.6968 3433 SER433 N −15.8397 −16.141 −0.2841 3434 SER433 CA −15.2145−17.355 0.2126 3435 SER433 C −13.7172 −17.2481 0.2387 3436 SER433 O−13.1583 −16.9925 1.2919 3437 SER433 CB −15.6441 −18.5942 −0.5969 3438SER433 OG −15.599 −18.3117 −1.9987 3439 SER434 N −13.0628 −17.4649−0.9199 3440 SER434 CA −11.6091 −17.4594 −0.9335 3441 SER434 C −11.0874−16.1277 −0.4781 3442 SER434 O −10.2951 −16.0703 0.4479 3443 SER434 CB−11.1082 −17.7736 −2.3567 3444 SER434 OG −11.7692 −16.9353 −3.3093 3445LEU435 N −11.5575 −15.0507 −1.1328 3446 LEU435 CA −11.1601 −13.7259−0.6947 3447 LEU435 C −11.8117 −13.4319 0.6284 3448 LEU435 O −11.2044−12.7876 1.4682 3449 LEU435 CB −11.6284 −12.7274 −1.7707 3450 LEU435 CG−10.9874 −13.0944 −3.1253 3451 LEU435 CD1 −11.6377 −12.2835 −4.2611 3452LEU435 CD2 −9.4715 −12.8205 −3.0924 3453 GLY436 N −13.0511 −13.93010.8141 3454 GLY436 CA −13.7463 −13.6783 2.0645 3455 GLY436 C −13.1259−14.3795 3.2413 3456 GLY436 O −13.5195 −14.0929 4.3606 3457 SER437 N−12.1546 −15.2873 3.0096 3458 SER437 CA −11.4627 −15.8787 4.1435 3459SER437 C −10.6472 −14.7866 4.7693 3460 SER437 O −10.7271 −14.5724 5.96783461 SER437 CB −10.5753 −17.0588 3.6991 3462 SER437 OG −9.4322 −16.6052.9694 3463 ARG438 N −9.8934 −14.0746 3.9088 3464 ARG438 CA −9.3036−12.8255 4.3441 3465 ARG438 C −10.3339 −11.7506 4.1274 3466 ARG438 O−10.0357 −10.6992 3.5855 3467 ARG438 CB −8.0394 −12.5637 3.5059 3468ARG438 CG −6.799 −12.675 4.4085 3469 ARG438 CD −5.537 −12.5279 3.54263470 ARG438 NE −5.2563 −13.78 2.8628 3471 ARG438 CZ −5.3165 −13.87471.5657 3472 ARG438 NH1 −5.0562 −15.0148 0.9988 3473 ARG438 NH2 −5.633−12.8539 0.824 3474 GLN439 N −11.5724 −12.058 4.5619 3475 GLN439 CA−12.6718 −11.1177 4.4405 3476 GLN439 C −12.5563 −9.9071 5.3258 3477GLN439 O −11.6003 −9.1648 5.1923 3478 GLN439 CB −13.1032 −10.8149 2.99123479 GLN439 CG −12.3806 −9.5842 2.4087 3480 GLN439 CD −11.8279 −9.91461.0526 3481 GLN439 OE1 −10.6294 −9.8213 0.8458 3482 GLN439 NE2 −12.7032−10.308 0.1101 3483 PRO440 N −13.5257 −9.6736 6.2322 3484 PRO440 CA−13.5102 −8.454 7.0132 3485 PRO440 C −12.5963 −8.5337 8.2091 3486 PRO440O −13.0331 −8.9057 9.2865 3487 PRO440 CB −14.9903 −8.3729 7.4378 3488PRO440 CG −15.6183 −9.7669 7.2251 3489 PRO440 CD −14.6348 −10.59246.3754 3490 PHE441 N −11.313 −8.154 8.0356 3491 PHE441 CA −10.4329−8.1481 9.1921 3492 PHE441 C −9.3135 −7.1499 9.079 3493 PHE441 O −9.307−6.1968 9.8411 3494 PHE441 CB −9.9065 −9.5694 9.4543 3495 PHE441 CG−8.8765 −9.5947 10.5777 3496 PHE441 CD1 −9.2572 −9.3263 11.8948 3497PHE441 CD2 −7.5447 −9.8939 10.2826 3498 PHE441 CE1 −8.3156 −9.405412.9238 3499 PHE441 CE2 −6.598 −9.9539 11.3085 3500 PHE441 CZ −6.9871−9.7246 12.6308 3501 PHE442 N −8.3614 −7.3754 8.1492 3502 PHE442 CA−7.2088 −6.4892 8.0549 3503 PHE442 C −7.6206 −5.0488 7.9371 3504 PHE442O −7.2474 −4.2463 8.7776 3505 PHE442 CB −6.3572 −6.8537 6.8211 3506PHE442 CG −5.5543 −8.1401 7.0126 3507 PHE442 CD1 −5.6384 −8.883 8.19343508 PHE442 CD2 −4.7192 −8.579 5.9815 3509 PHE442 CE1 −4.9145 −10.07058.3302 3510 PHE442 CE2 −4.0186 −9.7817 6.1024 3511 PHE442 CZ −4.1198−10.531 7.2771 3512 SER443 N −8.4024 −4.7357 6.8855 3513 SER443 CA−8.8934 −3.3779 6.7157 3514 SER443 C −7.8833 −2.4435 6.119 3515 SER443 O−6.7567 −2.3707 6.5828 3516 SER443 CB −9.561 −2.7648 7.9634 3517 SER443OG −10.3106 −3.7529 8.6741 3518 LEU444 N −8.3187 −1.7202 5.0706 3519LEU444 CA −7.4518 −0.7066 4.4982 3520 LEU444 C −7.9637 0.6451 4.91173521 LEU444 O −9.1482 0.7865 5.1681 3522 LEU444 CB −7.4563 −0.83422.9623 3523 LEU444 CG −6.0403 −0.638 2.3877 3524 LEU444 CD1 −6.0698−0.9038 0.8713 3525 LEU444 CD2 −5.5569 0.8034 2.6349 3526 GLU445 N−7.0692 1.6498 4.9992 3527 GLU445 CA −7.5102 2.9652 5.4428 3528 GLU445 C−8.0475 2.9437 6.8497 3529 GLU445 O −8.5261 3.9614 7.3251 3530 GLU445 CB−8.5437 3.576 4.4742 3531 GLU445 CG −7.8604 3.8923 3.1312 3532 GLU445 CD−8.464 3.0583 2.0392 3533 GLU445 OE1 −9.0241 3.6551 1.0816 3534 GLU445OE2 −8.378 1.8052 2.1324 3535 ALA446 N −7.9729 1.7703 7.5121 3536 ALA446CA −8.5063 1.6526 8.8598 3537 ALA446 C −8.4942 0.2083 9.2776 3538 ALA446O −7.7962 −0.5946 8.679 3539 ALA446 CB −9.9677 2.1342 8.881 3540 CYS447N −9.2914 −0.1215 10.3135 3541 CYS447 CA −9.3604 −1.5071 10.7405 3542CYS447 C −10.7781 −1.9128 11.0309 3543 CYS447 O −11.6868 −1.1019 10.94393544 CYS447 CB −8.4782 −1.7215 11.9838 3545 CYS447 SG −6.7617 −1.884911.4129 3546 GLN448 N −10.9572 −3.2019 11.3776 3547 GLN448 CA −12.2997−3.6777 11.6568 3548 GLN448 C −12.3559 −4.3087 13.021 3549 GLN448 O−11.3584 −4.3311 13.7248 3550 GLN448 CB −12.6929 −4.7019 10.576 3551GLN448 CG −14.2214 −4.8842 10.5841 3552 GLN448 CD −14.5629 −6.344610.5964 3553 GLN448 OE1 −15.2139 −6.8123 9.6784 3554 GLN448 NE2 −14.1306−7.0806 11.6362 3555 GLY449 N −13.5408 −4.8271 13.3986 3556 GLY449 CA−13.6521 −5.4298 14.7135 3557 GLY449 C −14.9421 −6.1864 14.8275 3558GLY449 O −15.9341 −5.6338 15.2739 3559 ILE450 N −14.9096 −7.4713 14.42653560 ILE450 CA −16.0878 −8.305 14.5969 3561 ILE450 C −16.2627 −8.660116.0492 3562 ILE450 O −15.3354 −8.5252 16.8306 3563 ILE450 CB −15.9326−9.5842 13.7436 3564 ILE450 CG1 −16.9995 −10.6372 14.115 3565 ILE450 CG2−14.5118 −10.1605 13.8955 3566 ILE450 CD1 −16.6492 −12.0235 13.5415 3567LEU451 N −17.4745 −9.1269 16.4041 3568 LEU451 CA −17.6792 −9.627717.7517 3569 LEU451 C −18.6535 −10.7714 17.7146 3570 LEU451 O −19.1634−11.1042 16.6561 3571 LEU451 CB −18.2011 −8.5351 18.7037 3572 LEU451 CG−17.1039 −7.5035 19.0253 3573 LEU451 CD1 −17.0986 −6.4004 17.9498 3574LEU451 CD2 −17.4062 −6.8647 20.3934 3575 ALA452 N −18.914 −11.385518.8851 3576 ALA452 CA −19.811 −12.5274 18.8843 3577 ALA452 C −20.4078−12.7757 20.2417 3578 ALA452 O −20.1361 −12.0456 21.1807 3579 ALA452 CB−19.0467 −13.7711 18.3944 3580 LEU453 N −21.2402 −13.8306 20.3318 3581LEU453 CA −21.8105 −14.1856 21.6185 3582 LEU453 C −22.2269 −15.625921.5694 3583 LEU453 O −22.9677 −16.0119 20.6803 3584 LEU453 CB −23.0163−13.2979 21.9873 3585 LEU453 CG −23.0743 −13.0848 23.5134 3586 LEU453CD1 −24.2561 −12.1686 23.8721 3587 LEU453 CD2 −23.2629 −14.4276 24.24043588 LEU454 N −21.7179 −16.4119 22.538 3589 LEU454 CA −22.0031 −17.837422.5715 3590 LEU454 C −21.7649 −18.549 21.2663 3591 LEU454 O −20.6473−18.9583 20.9962 3592 LEU454 CB −23.3365 −18.2012 23.258 3593 LEU454 CG−24.5355 −17.7682 22.3939 3594 LEU454 CD1 −25.5516 −18.9204 22.3016 3595LEU454 CD2 −25.1978 −16.5096 22.9825 3596 ASP455 N −22.8361 −18.704920.4641 3597 ASP455 CA −22.7111 −19.4269 19.2122 3598 ASP455 C −23.9693−19.2033 18.4191 3599 ASP455 O −24.6883 −20.1354 18.0977 3600 ASP455 CB−22.4065 −20.918 19.468 3601 ASP455 CG −23.4556 −21.5473 20.3398 3602ASP455 OD1 −23.4766 −21.2391 21.5615 3603 ASP455 OD2 −24.2562 −22.359519.8046 3604 LEU456 N −24.2256 −17.9188 18.1067 3605 LEU456 CA −25.4488−17.5741 17.4012 3606 LEU456 C −25.4177 −18.0274 15.967 3607 LEU456 O−24.4079 −18.5308 15.5021 3608 LEU456 CB −25.7189 −16.0568 17.4969 3609LEU456 CG −25.2245 −15.4804 18.8418 3610 LEU456 CD1 −25.6259 −14.001918.9853 3611 LEU456 CD2 −25.8085 −16.2725 20.0232 3612 ASN457 N −26.5491−17.8556 15.2576 3613 ASN457 CA −26.6035 −18.3511 13.894 3614 ASN457 C−26.5348 −17.2364 12.8876 3615 ASN457 O −26.5467 −17.5147 11.6988 3616ASN457 CB −27.8819 −19.1935 13.7129 3617 ASN457 CG −27.8633 −19.925512.4013 3618 ASN457 OD1 −28.7526 −19.734 11.5886 3619 ASN457 ND2−26.8431 −20.773 12.1774 3620 ALA458 N −26.4576 −15.9721 13.3534 3621ALA458 CA −26.3819 −14.8723 12.4045 3622 ALA458 C −25.196 −15.073911.5085 3623 ALA458 O −25.3624 −15.2125 10.3077 3624 ALA458 CB −26.2713−13.5328 13.1531 3625 SER459 N −23.9936 −15.1232 12.1096 3626 SER459 CA−22.8526 −15.5511 11.3264 3627 SER459 C −22.8258 −17.0523 11.3748 3628SER459 O −22.1743 −17.633 12.2275 3629 SER459 CB −21.5611 −14.900411.8587 3630 SER459 OG −21.5207 −14.9551 13.2861 3631 GLY460 N −23.5545−17.6907 10.4395 3632 GLY460 CA −23.5731 −19.1412 10.4551 3633 GLY460 C−24.3729 −19.7103 9.3209 3634 GLY460 O −24.3669 −20.9183 9.1474 3635THR461 N −25.0651 −18.8512 8.5476 3636 THR461 CA −25.8776 −19.38987.4721 3637 THR461 C −25.8192 −18.4964 6.2644 3638 THR461 O −25.8752−17.2885 6.42 3639 THR461 CB −27.332 −19.5874 7.9424 3640 THR461 OG1−27.3496 −20.3788 9.1335 3641 THR461 CG2 −28.1507 −20.3002 6.8495 3642MET462 N −25.7015 −19.054 5.0422 3643 MET462 CA −25.6916 −20.5014 4.91153644 MET462 C −24.3543 −21.0862 5.2649 3645 MET462 O −24.2089 −21.60136.3617 3646 MET462 CB −26.1399 −20.9827 3.5177 3647 MET462 CG −27.1019−19.9726 2.8654 3648 MET462 SD −26.0964 −18.9071 1.7923 3649 MET462 CE−26.8403 −17.3145 2.2498 3650 SER463 N −23.3742 −21.0232 4.3405 3651SER463 CA −22.0933 −21.625 4.6622 3652 SER463 C −20.9364 −21.0807 3.8723653 SER463 O −21.1103 −20.3086 2.9427 3654 SER463 CB −22.1673 −23.15394.4857 3655 SER463 OG −22.2279 −23.478 3.0943 3656 ILE464 N −19.7303−21.5325 4.2705 3657 ILE464 CA −18.5404 −21.2112 3.5042 3658 ILE464 C−18.6579 −21.8491 2.1499 3659 ILE464 O −18.1781 −21.2911 1.1766 3660ILE464 CB −17.2947 −21.7419 4.2472 3661 ILE464 CG1 −16.0361 −21.52443.3814 3662 ILE464 CG2 −17.4593 −23.237 4.5853 3663 ILE464 CD1 −14.7779−22.0522 4.0959 3664 GLN465 N −19.3259 −23.0197 2.1078 3665 GLN465 CA−19.5831 −23.6643 0.8319 3666 GLN465 C −20.3295 −22.7182 −0.0651 3667GLN465 O −20.1229 −22.7443 −1.2678 3668 GLN465 CB −20.444 −24.91651.0837 3669 GLN465 CG −19.7163 −25.8538 2.0665 3670 GLN465 CD −20.3202−25.7168 3.434 3671 GLN465 OE1 −19.6984 −25.1588 4.3229 3672 GLN465 NE2−21.5492 −26.2327 3.6147 3673 GLU466 N −21.1788 −21.8589 0.5345 3674GLU466 CA −21.8194 −20.8295 −0.2637 3675 GLU466 C −20.8655 −19.7025−0.5599 3676 GLU466 O −21.2575 −18.5461 −0.5642 3677 GLU466 CB −23.1093−20.3379 0.4195 3678 GLU466 CG −24.3071 −21.0682 −0.2163 3679 GLU466 CD−25.0033 −21.9491 0.7812 3680 GLU466 OE1 −26.2538 −22.0654 0.6838 3681GLU466 OE2 −24.3149 −22.5175 1.6704 3682 PHE467 N −19.5915 −20.0647−0.8145 3683 PHE467 CA −18.6057 −19.0773 −1.2217 3684 PHE467 C −18.4992−17.9348 −0.2519 3685 PHE467 O −18.7028 −16.7913 −0.6279 3686 PHE467 CB−18.8775 −18.6158 −2.6665 3687 PHE467 CG −18.8007 −19.8306 −3.5833 3688PHE467 CD1 −19.9284 −20.6341 −3.7711 3689 PHE467 CD2 −17.6019 −20.1412−4.2302 3690 PHE467 CE1 −19.8486 −21.7681 −4.5831 3691 PHE467 CE2−17.5246 −21.272 −5.0471 3692 PHE467 CZ −18.6458 −22.0883 −5.2185 3693ARG468 N −18.1687 −18.2613 1.013 3694 ARG468 CA −18.1013 −17.2092 2.00993695 ARG468 C −17.3189 −17.6414 3.219 3696 ARG468 O −17.2096 −18.83093.4638 3697 ARG468 CB −19.5391 −16.8 2.3619 3698 ARG468 CG −19.9077−15.5407 1.5629 3699 ARG468 CD −21.3668 −15.6322 1.0869 3700 ARG468 NE−21.6434 −14.468 0.2675 3701 ARG468 CZ −22.7141 −13.7518 0.4496 3702ARG468 NH1 −22.9115 −12.7039 −0.2936 3703 ARG468 NH2 −23.5889 −14.06411.3599 3704 ASP469 N −16.745 −16.6834 3.9761 3705 ASP469 CA −15.8373−17.0925 5.037 3706 ASP469 C −16.05 −16.3696 6.3396 3707 ASP469 O−16.7493 −15.3712 6.3862 3708 ASP469 CB −14.3729 −16.9777 4.571 3709ASP469 CG −13.9302 −18.2086 3.8302 3710 ASP469 OD1 −12.8642 −18.76714.199 3711 ASP469 OD2 −14.6368 −18.6322 2.8782 3712 LEU470 N −15.4321−16.918 7.4061 3713 LEU470 CA −15.6036 −16.3798 8.7478 3714 LEU470 C−14.9378 −15.0526 8.9918 3715 LEU470 O −14.3951 −14.435 8.0894 3716LEU470 CB −17.0618 −16.4526 9.2636 3717 LEU470 CG −17.5558 −15.10049.8279 3718 LEU470 CD1 −17.8156 −14.1091 8.6845 3719 LEU470 CD2 −18.8011−15.2192 10.7218 3720 TRP471 N −15.0438 −14.6356 10.2697 3721 TRP471 CA−14.7445 −13.2703 10.6552 3722 TRP471 C −13.4505 −13.1482 11.3955 3723TRP471 O −12.402 −13.4452 10.8476 3724 TRP471 CB −15.0216 −12.17789.6047 3725 TRP471 CG −15.8341 −11.0716 10.2105 3726 TRP471 CD1 −15.4677−9.7868 10.3272 3727 TRP471 CD2 −17.2153 −11.191 10.7969 3728 TRP471 NE1−16.4268 −9.0953 10.8891 3729 TRP471 CE2 −17.4766 −9.8896 11.18 3730TRP471 CE3 −18.1224 −12.2308 10.9936 3731 TRP471 CZ2 −18.6801 −9.530211.7862 3732 TRP471 CZ3 −19.3127 −11.8885 11.647 3733 TRP471 CH2−19.5898 −10.5671 12.0256 3734 LYS472 N −13.5592 −12.7134 12.6662 3735LYS472 CA −12.3709 −12.5281 13.4815 3736 LYS472 C −11.6523 −11.265213.091 3737 LYS472 O −11.6137 −10.9442 11.9143 3738 LYS472 CB −11.4541−13.7677 13.4882 3739 LYS472 CG −10.8259 −13.9547 14.8805 3740 LYS472 CD−9.2936 −13.8414 14.7855 3741 LYS472 CE −8.8943 −12.4087 14.3892 3742LYS472 NZ −7.4446 −12.2088 14.5154 3743 GLN473 N −11.084 −10.505414.0493 3744 GLN473 CA −11.1305 −10.9221 15.4406 3745 GLN473 C −12.4414−10.5471 16.0684 3746 GLN473 O −12.6715 −9.3793 16.3395 3747 GLN473 CB−9.9771 −10.2204 16.1819 3748 GLN473 CG −9.5473 −11.0699 17.3916 3749GLN473 CD −8.0631 −11.2939 17.3549 3750 GLN473 OE1 −7.6174 −12.427517.289 3751 GLN473 NE2 −7.2768 −10.2035 17.398 3752 LEU474 N −13.3061−11.5517 16.3078 3753 LEU474 CA −14.5666 −11.2333 16.9532 3754 LEU474 C−14.5097 −11.4497 18.434 3755 LEU474 O −13.7945 −12.319 18.9057 3756LEU474 CB −15.7761 −11.9997 16.3893 3757 LEU474 CG −15.5066 −13.514416.358 3758 LEU474 CD1 −16.8462 −14.2616 16.2372 3759 LEU474 CD2−14.6256 −13.8496 15.1415 3760 LYS475 N −15.2938 −10.6362 19.1639 3761LYS475 CA −15.3764 −10.8467 20.594 3762 LYS475 C −16.4623 −11.853420.8451 3763 LYS475 O −17.5127 −11.5008 21.3576 3764 LYS475 CB −15.6919−9.5072 21.2876 3765 LYS475 CG −14.3884 −8.7313 21.5514 3766 LYS475 CD−14.1716 −7.6716 20.455 3767 LYS475 CE −13.5279 −8.3139 19.2124 3768LYS475 NZ −13.2432 −7.2662 18.2213 3769 LEU476 N −16.2048 −13.127420.4849 3770 LEU476 CA −17.2074 −14.1368 20.7723 3771 LEU476 C −17.3598−14.2772 22.2538 3772 LEU476 O −16.3956 −14.5589 22.9461 3773 LEU476 CB−16.9055 −15.5044 20.1284 3774 LEU476 CG −17.8622 −16.6139 20.6251 3775LEU476 CD1 −17.4057 −17.2144 21.9679 3776 LEU476 CD2 −19.3105 −16.105520.7499 3777 SER477 N −18.6041 −14.0714 22.7157 3778 SER477 CA −18.8438−14.1521 24.1395 3779 SER477 C −18.8661 −15.576 24.6074 3780 SER477 O−19.5043 −16.4165 23.9951 3781 SER477 CB −20.1921 −13.484 24.4527 3782SER477 OG −20.1822 −12.1688 23.8963 3783 GLN478 N −18.1605 −15.837525.7225 3784 GLN478 CA −18.2964 −17.1389 26.3477 3785 GLN478 C −19.6603−17.184 26.9769 3786 GLN478 O −20.2313 −18.2527 27.1221 3787 GLN478 CB−17.1909 −17.314 27.4078 3788 GLN478 CG −15.7783 −17.1447 26.8039 3789GLN478 CD −15.6896 −17.5401 25.3558 3790 GLN478 OE1 −15.394 −16.706624.5156 3791 GLN478 NE2 −15.947 −18.8243 25.0503 3792 LYS479 N −20.1891−15.989 27.3126 3793 LYS479 CA −21.5695 −15.9001 27.7579 3794 LYS479 C−22.518 −16.3006 26.6601 3795 LYS479 O −22.093 −16.6819 25.5809 3796LYS479 CB −21.9024 −14.4684 28.2308 3797 LYS479 CG −21.1271 −13.406427.4257 3798 LYS479 CD −19.8093 −13.0884 28.158 3799 LYS479 CE −18.6074−13.3679 27.239 3800 LYS479 NZ −18.3823 −12.2219 26.3474 3801 VAL480 N−23.8296 −16.2161 26.9525 3802 VAL480 CA −24.8025 −16.6796 25.9789 3803VAL480 C −25.9383 −15.6936 25.8977 3804 VAL480 O −25.7435 −14.523526.1871 3805 VAL480 CB −25.2777 −18.0865 26.4048 3806 VAL480 CG1−26.1324 −18.7344 25.2995 3807 VAL480 CG2 −24.0673 −18.9988 26.6815 3808PHE481 N −27.1361 −16.1703 25.5011 3809 PHE481 CA −28.2686 −15.268825.3871 3810 PHE481 C −28.5777 −14.6108 26.6995 3811 PHE481 O −28.5512−15.2663 27.7289 3812 PHE481 CB −29.5093 −16.0464 24.9095 3813 PHE481 CG−30.6455 −15.1135 24.4987 3814 PHE481 CD1 −30.405 −13.7648 24.2231 3815PHE481 CD2 −31.9427 −15.622 24.3969 3816 PHE481 CE1 −31.4575 −12.92923.8457 3817 PHE481 CE2 −32.9913 −14.7928 23.9908 3818 PHE481 CZ−32.7463 −13.4484 23.7008 3819 HIS482 N −28.8647 −13.2949 26.6425 3820HIS482 CA −29.1666 −12.5712 27.8657 3821 HIS482 C −28.0142 −12.668328.827 3822 HIS482 O −28.2101 −12.9102 30.0076 3823 HIS482 CB −30.4761−13.0913 28.4932 3824 HIS482 CG −31.6535 −12.8039 27.6078 3825 HIS482ND1 −32.6249 −13.674 27.4567 3826 HIS482 CD2 −31.8465 −11.6717 26.90443827 HIS482 CE1 −33.4945 −13.1446 26.6562 3828 HIS482 NE2 −33.1102−12.0025 26.3046 3829 LYS483 N −26.7884 −12.4904 28.299 3830 LYS483 CA−25.6324 −12.6329 29.1636 3831 LYS483 C −24.6189 −11.5883 28.8083 3832LYS483 O −24.1628 −11.5435 27.6766 3833 LYS483 CB −25.0266 −14.044229.0363 3834 LYS483 CG −26.1005 −15.0942 29.3754 3835 LYS483 CD −25.5128−16.5103 29.2494 3836 LYS483 CE −26.6608 −17.5359 29.2614 3837 LYS483 NZ−26.1029 −18.8953 29.2265 3838 GLN484 N −24.2798 −10.7423 29.8014 3839GLN484 CA −23.3352 −9.6715 29.5338 3840 GLN484 C −22.0236 −10.225229.0607 3841 GLN484 O −21.606 −11.2796 29.5116 3842 GLN484 CB −23.0998−8.8037 30.7839 3843 GLN484 CG −24.4469 −8.3374 31.367 3844 GLN484 CD−24.5187 −8.7226 32.8162 3845 GLN484 OE1 −25.3451 −9.5393 33.1882 3846GLN484 NE2 −23.647 −8.1311 33.6529 3847 ASP485 N −21.3878 −9.501 28.12253848 ASP485 CA −20.1652 −10.0221 27.5422 3849 ASP485 C −18.9649 −9.699728.3851 3850 ASP485 O −19.0926 −9.0979 29.4395 3851 ASP485 CB −20.0082−9.4693 26.1131 3852 ASP485 CG −21.1396 −9.9172 25.2301 3853 ASP485 OD1−21.3088 −9.3015 24.1442 3854 ASP485 OD2 −21.8672 −10.8755 25.6106 3855ARG486 N −17.7821 −10.1222 27.8996 3856 ARG486 CA −16.5738 −9.91528.6768 3857 ARG486 C −15.3703 −10.2696 27.8524 3858 ARG486 O −15.4489−11.148 27.0082 3859 ARG486 CB −16.5904 −10.7773 29.9554 3860 ARG486 CG−16.9669 −12.2259 29.5918 3861 ARG486 CD −16.9507 −13.1163 30.8459 3862ARG486 NE −16.4438 −14.423 30.4703 3863 ARG486 CZ −15.4104 −14.94131.0681 3864 ARG486 NH1 −14.9709 −16.1035 30.6875 3865 ARG486 NH2−14.8105 −14.3159 32.0383 3866 GLY487 N −14.2495 −9.5658 28.1093 3867GLY487 CA −13.0384 −9.848 27.3574 3868 GLY487 C −12.5891 −11.255427.6213 3869 GLY487 O −12.254 −11.9666 26.6877 3870 SER488 N −12.6035−11.6512 28.91 3871 SER488 CA −12.2732 −13.0265 29.2428 3872 SER488 C−13.2361 −13.9456 28.5477 3873 SER488 O −12.8507 −15.0267 28.1324 3874SER488 CB −12.4306 −13.2055 30.7638 3875 SER488 OG −11.5098 −12.353731.45 3876 GLY489 N −14.4953 −13.4891 28.4056 3877 GLY489 CA −15.4568−14.2927 27.6778 3878 GLY489 C −15.3005 −14.0949 26.1974 3879 GLY489 O−16.3031 −13.9565 25.519 3880 TYR490 N −14.0514 −14.0902 25.6914 3881TYR490 CA −13.8643 −13.9993 24.2515 3882 TYR490 C −12.8647 −15.031123.8046 3883 TYR490 O −12.3326 −15.7491 24.6367 3884 TYR490 CB −13.3901−12.5919 23.8351 3885 TYR490 CG −14.4754 −11.5416 24.0603 3886 TYR490CD1 −14.1148 −10.2607 24.4856 3887 TYR490 CD2 −15.8226 −11.8443 23.84633888 TYR490 CE1 −15.0993 −9.2996 24.7292 3889 TYR490 CE2 −16.8117−10.8964 24.1178 3890 TYR490 CZ −16.4483 −9.6229 24.562 3891 TYR490 OH−17.4285 −8.6779 24.8375 3892 LEU491 N −12.6034 −15.1172 22.4831 3893LEU491 CA −11.6638 −16.129 22.0245 3894 LEU491 C −11.252 −15.945620.5902 3895 LEU491 O −12.0934 −15.9191 19.7064 3896 LEU491 CB −12.1723−17.5602 22.3026 3897 LEU491 CG −13.3597 −17.9456 21.3964 3898 LEU491CD1 −13.8683 −19.3391 21.8069 3899 LEU491 CD2 −14.5076 −16.9305 21.54163900 ASN492 N −9.9258 −15.8234 20.3828 3901 ASN492 CA −9.4009 −15.675819.0336 3902 ASN492 C −7.9853 −16.1958 19.0018 3903 ASN492 O −7.4131−16.4041 20.0593 3904 ASN492 CB −9.4086 −14.1987 18.5951 3905 ASN492 CG−10.7986 −13.6329 18.5686 3906 ASN492 OD1 −11.1194 −12.7871 19.3868 3907ASN492 ND2 −11.639 −14.0921 17.6246 3908 TRP493 N −7.3666 −16.440517.8269 3909 TRP493 CA −8.033 −16.1681 16.5662 3910 TRP493 C −6.9975−15.7789 15.5421 3911 TRP493 O −6.647 −14.6114 15.4916 3912 TRP493 CB−8.8914 −17.3508 16.0732 3913 TRP493 CG −9.8756 −17.8632 17.0855 3914TRP493 CD1 −11.2065 −17.7072 17.0457 3915 TRP493 CD2 −9.5666 −18.648418.3321 3916 TRP493 NE1 −11.7642 −18.2709 18.0878 3917 TRP493 CE2−10.8239 −18.8265 18.8777 3918 TRP493 CE3 −8.4064 −19.1398 18.9289 3919TRP493 CZ2 −11.0102 −19.4955 20.0863 3920 TRP493 CZ3 −8.5843 −19.810720.1447 3921 TRP493 CH2 −9.8554 −19.9779 20.7139 3922 GLU494 N −6.4974−16.7269 14.7194 3923 GLU494 CA −5.4994 −16.3438 13.7304 3924 GLU494 C−5.5057 −17.2435 12.5255 3925 GLU494 O −5.0583 −18.3768 12.5985 3926GLU494 CB −4.0923 −16.2476 14.3499 3927 GLU494 CG −3.7763 −14.782514.7135 3928 GLU494 CD −4.102 −13.8467 13.5824 3929 GLU494 OE1 −4.7641−12.8096 13.8511 3930 GLU494 OE2 −3.7091 −14.1469 12.4229 3931 GLN495 N−6.0364 −16.7035 11.4097 3932 GLN495 CA −6.304 −17.5166 10.2357 3933GLN495 C −7.2692 −16.8182 9.3166 3934 GLN495 O −7.338 −15.5996 9.32543935 GLN495 CB −5.0369 −17.9776 9.493 3936 GLN495 CG −4.8209 −19.48769.7211 3937 GLN495 CD −6.0634 −20.2775 9.4157 3938 GLN495 OE1 −6.5357−21.0175 10.2627 3939 GLN495 NE2 −6.6081 −20.1267 8.195 3940 LEU496 N−8.0384 −17.6007 8.5327 3941 LEU496 CA −9.0582 −16.9857 7.6964 3942LEU496 C −10.3289 −17.7858 7.7509 3943 LEU496 O −10.2675 −18.9972 7.88423944 LEU496 CB −8.595 −16.8349 6.2352 3945 LEU496 CG −7.3993 −15.86846.14 3946 LEU496 CD1 −6.9522 −15.7636 4.6704 3947 LEU496 CD2 −7.801−14.4732 6.6533 3948 HIS497 N −11.4883 −17.0996 7.6701 3949 HIS497 CA−12.7528 −17.7812 7.9109 3950 HIS497 C −12.8025 −18.1945 9.3541 3951HIS497 O −12.5097 −19.3358 9.6708 3952 HIS497 CB −12.9863 −18.99536.9865 3953 HIS497 CG −14.3988 −19.4985 7.1136 3954 HIS497 ND1 −14.9898−19.6536 8.2769 3955 HIS497 CD2 −15.1943 −19.8347 6.0807 3956 HIS497 CE1−16.1873 −20.0931 8.0516 3957 HIS497 NE2 −16.3672 −20.2202 6.8162 3958ALA498 N −13.1814 −17.2429 10.2272 3959 ALA498 CA −13.1595 −17.51511.6542 3960 ALA498 C −14.0791 −18.6291 12.0738 3961 ALA498 O −14.7686−19.2061 11.248 3962 ALA498 CB −13.566 −16.215 12.3713 3963 ALA499 N−14.0686 −18.913 13.3931 3964 ALA499 CA −14.9678 −19.9106 13.9554 3965ALA499 C −14.5838 −20.2116 15.3775 3966 ALA499 O −13.7636 −19.516815.957 3967 ALA499 CB −14.9691 −21.2336 13.1688 3968 MET500 N −15.1933−21.276 15.9393 3969 MET500 CA −14.9391 −21.6046 17.3319 3970 MET500 C−15.426 −20.4754 18.1944 3971 MET500 O −14.7312 −20.0207 19.0888 3972MET500 CB −13.4583 −21.9532 17.5791 3973 MET500 CG −13.3701 −22.975918.7261 3974 MET500 SD −12.7965 −22.1232 20.2218 3975 MET500 CE −12.6583−23.5784 21.2996 3976 ARG501 N −16.6523 −20.0177 17.8795 3977 ARG501 CA−17.1808 −18.8494 18.5571 3978 ARG501 C −18.6731 −18.8256 18.3681 3979ARG501 O −19.2805 −19.8657 18.1664 3980 ARG501 CB −16.5623 −17.612817.8727 3981 ARG501 CG −15.1516 −17.3417 18.4288 3982 ARG501 CD −14.67−15.9651 17.9376 3983 ARG501 NE −13.573 −16.1153 16.9993 3984 ARG501 CZ−13.7607 −16.5824 15.7985 3985 ARG501 NH1 −12.7489 −16.6829 14.9893 3986ARG501 NH2 −14.9398 −16.9495 15.3919 3987 GLU502 N −19.2645 −17.614918.4157 3988 GLU502 CA −20.6847 −17.4904 18.1419 3989 GLU502 C −20.9358−18.0166 16.7604 3990 GLU502 O −21.946 −18.6548 16.5213 3991 GLU502 CB−21.034 −15.992 18.1823 3992 GLU502 CG −22.4733 −15.7624 17.6882 3993GLU502 CD −22.4706 −15.3281 16.2512 3994 GLU502 OE1 −23.0568 −16.06215.4118 3995 GLU502 OE2 −21.886 −14.2506 15.958 3996 ALA503 N −19.9852−17.7542 15.8483 3997 ALA503 CA −20.1081 −18.3485 14.5355 3998 ALA503 C−19.1648 −19.5142 14.4331 3999 ALA503 O −18.1384 −19.5231 15.095 4000ALA503 CB −19.7811 −17.2624 13.5006 4001 GLY504 N −19.5234 −20.516913.6078 4002 GLY504 CA −18.6468 −21.6697 13.4978 4003 GLY504 C −19.42−22.9573 13.4308 4004 GLY504 O −20.6152 −22.9529 13.1838 4005 ARG505 N−18.7084 −24.0801 13.6488 4006 ARG505 CA −19.3631 −25.3686 13.5231 4007ARG505 C −20.074 −25.7173 14.7992 4008 ARG505 O −19.5131 −26.377115.6597 4009 ARG505 CB −18.3277 −26.4418 13.1353 4010 ARG505 CG −19.0675−27.7271 12.72 4011 ARG505 CD −18.0544 −28.8431 12.4085 4012 ARG505 NE−17.7481 −28.8403 10.9898 4013 ARG505 CZ −16.5174 −28.8541 10.5667 4014ARG505 NH1 −16.2831 −28.843 9.2882 4015 ARG505 NH2 −15.5188 −28.875111.4002 4016 HIS506 N −21.3392 −25.2707 14.9051 4017 HIS506 CA −22.0972−25.5838 16.103 4018 HIS506 C −23.5704 −25.4112 15.8649 4019 HIS506 O−23.9709 −24.8666 14.8481 4020 HIS506 CB −21.6667 −24.6447 17.2475 4021HIS506 CG −21.8326 −23.2214 16.8002 4022 HIS506 ND1 −20.8661 −22.567316.199 4023 HIS506 CD2 −22.9533 −22.4907 16.9519 4024 HIS506 CE1−21.3155 −21.3829 15.9318 4025 HIS506 NE2 −22.4991 −21.2742 16.3357 4026ARG507 N −24.3781 −25.8767 16.8381 4027 ARG507 CA −25.8061 −25.630716.7529 4028 ARG507 C −26.0315 −24.1818 17.0795 4029 ARG507 O −26.2062−23.8255 18.2343 4030 ARG507 CB −26.5313 −26.5302 17.7719 4031 ARG507 CG−26.5793 −27.9768 17.2479 4032 ARG507 CD −27.4735 −28.8176 18.177 4033ARG507 NE −27.5376 −30.1818 17.6859 4034 ARG507 CZ −27.1634 −31.182318.4298 4035 ARG507 NH1 −27.2354 −32.3908 17.9563 4036 ARG507 NH2−26.7191 −30.9953 19.6381 4037 LYS508 N −26.0164 −23.3389 16.03 4038LYS508 CA −26.1133 −21.911 16.2711 4039 LYS508 C −27.4339 −21.515816.8629 4040 LYS508 O −28.4323 −22.1882 16.6601 4041 LYS508 CB −25.8432−21.1072 14.9894 4042 LYS508 CG −24.5525 −21.6192 14.3297 4043 LYS508 CD−24.0293 −20.5702 13.3357 4044 LYS508 CE −22.737 −21.1133 12.7056 4045LYS508 NZ −23.0643 −22.1664 11.7337 4046 SER509 N −27.4122 −20.406317.6226 4047 SER509 CA −28.6287 −19.9826 18.289 4048 SER509 C −29.2901−18.8631 17.5368 4049 SER509 O −28.8376 −18.4787 16.4708 4050 SER509 CB−28.2766 −19.5319 19.7192 4051 SER509 OG −27.1009 −18.72 19.6917 4052TRP510 N −30.3851 −18.3351 18.1135 4053 TRP510 CA −31.0793 −17.252517.4436 4054 TRP510 C −31.6712 −16.3401 18.4731 4055 TRP510 O −31.5666−15.1336 18.328 4056 TRP510 CB −32.1576 −17.8213 16.506 4057 TRP510 CG−31.5471 −17.9643 15.1431 4058 TRP510 CD1 −31.6121 −19.0333 14.3346 4059TRP510 CD2 −30.7387 −16.9143 14.4302 4060 TRP510 NE1 −30.9688 −18.787913.2198 4061 TRP510 CE2 −30.4479 −17.5436 13.235 4062 TRP510 CE3−30.3098 −15.6237 14.7371 4063 TRP510 CZ2 −29.7138 −16.9085 12.2337 4064TRP510 CZ3 −29.5691 −14.98 13.7383 4065 TRP510 CH2 −29.298 −15.600212.5096 4066 SER511 N −32.2648 −16.9216 19.5345 4067 SER511 CA −32.6247−16.0935 20.6714 4068 SER511 C −31.3371 −15.569 21.239 4069 SER511 O−31.1914 −14.3694 21.4039 4070 SER511 CB −33.3761 −16.9395 21.7181 4071SER511 OG −32.5848 −18.0604 22.1239 4072 CYS512 N −30.3893 −16.493121.4971 4073 CYS512 CA −29.054 −16.0688 21.8787 4074 CYS512 C −28.4923−15.2338 20.7654 4075 CYS512 O −27.8063 −14.2613 21.0336 4076 CYS512 CB−28.1483 −17.3054 22.0288 4077 CYS512 SG −28.9325 −18.5393 23.1075 4078GLY513 N −28.8125 −15.6145 19.5098 4079 GLY513 CA −28.404 −14.796518.3796 4080 GLY513 C −28.8091 −13.3673 18.6036 4081 GLY513 O −28.0983−12.4604 18.202 4082 HIS514 N −29.9612 −13.1856 19.2758 4083 HIS514 CA−30.3937 −11.8433 19.6082 4084 HIS514 C −29.7979 −11.4447 20.9308 4085HIS514 O −30.5143 −11.0878 21.8524 4086 HIS514 CB −31.9352 −11.814319.6557 4087 HIS514 CG −32.5477 −12.2504 18.3549 4088 HIS514 ND1−33.8314 −12.5101 18.2644 4089 HIS514 CD2 −31.8956 −12.4211 17.188 4090HIS514 CE1 −34.0697 −12.8611 17.0406 4091 HIS514 NE2 −33.0067 −12.831316.3739 4092 THR515 N −28.4557 −11.5005 21.0224 4093 THR515 CA −27.8194−11.0067 22.2293 4094 THR515 C −26.3531 −10.7594 22.0257 4095 THR515 O−25.697 −11.4658 21.2771 4096 THR515 CB −28.1139 −11.8416 23.4938 4097THR515 OG1 −27.8485 −11.0635 24.664 4098 THR515 CG2 −27.2537 −13.11923.5284 4099 ARG516 N −25.8659 −9.7201 22.7263 4100 ARG516 CA −24.4561−9.3847 22.6753 4101 ARG516 C −24.1989 −8.6831 23.9778 4102 ARG516 O−24.063 −7.4704 24.0149 4103 ARG516 CB −24.1919 −8.4614 21.4679 4104ARG516 CG −24.1308 −9.2883 20.1698 4105 ARG516 CD −22.8747 −10.178320.1863 4106 ARG516 NE −23.2084 −11.5089 19.7188 4107 ARG516 CZ −22.8227−11.9319 18.5506 4108 ARG516 NH1 −23.1216 −13.1413 18.1896 4109 ARG516NH2 −22.1438 −11.1823 17.7344 4110 ALA517 N −24.1631 −9.4895 25.05884111 ALA517 CA −24.1682 −8.9339 26.4027 4112 ALA517 C −25.5826 −8.554626.7308 4113 ALA517 O −26.1868 −9.1632 27.5994 4114 ALA517 CB −23.1864−7.7643 26.6139 4115 GLY518 N −26.1113 −7.5522 26.0026 4116 GLY518 CA−27.5232 −7.2444 26.1371 4117 GLY518 C −28.2821 −7.9608 25.0554 4118GLY518 O −27.716 −8.263 24.0173 4119 CYS519 N −29.5766 −8.2345 25.30954120 CYS519 CA −30.3711 −8.9519 24.3259 4121 CYS519 C −30.5352 −8.127523.0777 4122 CYS519 O −31.4697 −7.3499 22.9756 4123 CYS519 CB −31.7408−9.2363 24.9747 4124 CYS519 SG −32.8006 −10.1165 23.7893 4125 THR520 N−29.6209 −8.3119 22.1069 4126 THR520 CA −29.7507 −7.5622 20.8694 4127THR520 C −30.8517 −8.1256 20.0101 4128 THR520 O −31.5034 −9.0805 20.39944129 THR520 CB −28.394 −7.5683 20.1334 4130 THR520 OG1 −28.4296 −6.67819.0149 4131 THR520 CG2 −28.0242 −8.9858 19.6561 4132 LEU521 N −31.0527−7.5245 18.8205 4133 LEU521 CA −31.9831 −8.0999 17.8603 4134 LEU521 C−33.3511 −8.3288 18.4454 4135 LEU521 O −33.9048 −9.4088 18.3106 4136LEU521 CB −31.4089 −9.3888 17.2327 4137 LEU521 CG −29.882 −9.297917.0395 4138 LEU521 CD1 −29.3723 −10.6018 16.3999 4139 LEU521 CD2 −29.53−8.1117 16.1223 4140 ILE522 N −33.8974 −7.2926 19.1103 4141 ILE522 CA−35.2004 −7.4612 19.7307 4142 ILE522 C −35.9061 −6.1378 19.7926 4143ILE522 O −37.0732 −6.0582 19.4452 4144 ILE522 CB −35.056 −8.0071 21.16694145 ILE522 CG1 −34.1288 −7.105 22.0053 4146 ILE522 CG2 −34.5223 −9.451821.1494 4147 ILE522 CD1 −34.4094 −7.314 23.5048 4148 ARG523 N −35.1764−5.0996 20.246 4149 ARG523 CA −35.7994 −3.8012 20.4375 4150 ARG523 C−36.2561 −3.1922 19.1412 4151 ARG523 O −35.9804 −3.7237 18.0772 4152ARG523 CB −34.7842 −2.8678 21.1238 4153 ARG523 CG −34.2737 −3.523422.4214 4154 ARG523 CD −32.8623 −2.9969 22.7386 4155 ARG523 NE −32.2864−3.7696 23.8244 4156 ARG523 CZ −31.2373 −4.5175 23.6371 4157 ARG523 NH1−30.746 −5.1887 24.6364 4158 ARG523 NH2 −30.6715 −4.607 22.4691 4159GLN524 N −36.9758 −2.0581 19.2423 4160 GLN524 CA −37.4924 −1.447518.0298 4161 GLN524 C −37.3061 0.0429 18.0658 4162 GLN524 O −36.82460.5916 19.0436 4163 GLN524 CB −38.9918 −1.7647 17.8648 4164 GLN524 CG−39.2516 −3.2706 18.0637 4165 GLN524 CD −39.684 −3.5165 19.4805 4166GLN524 OE1 −38.9756 −4.1667 20.231 4167 GLN524 NE2 −40.8636 −2.993519.8606

TABLE V Atom No Residue Atom name x coord y coord z coord 1 LYS12 CA−26.781 19.61 −5.574 2 LYS12 C −27.853 18.529 −5.301 3 LYS12 N −27.30720.9 −5.159 4 LYS12 O −27.815 17.778 −4.324 5 LYS12 CB −25.462 19.387−4.819 6 LYS12 CG −24.469 18.447 −5.526 7 LYS12 CD −24.861 16.966 −5.5118 LYS12 CE −23.82 16.057 −6.165 9 LYS12 NZ −22.573 16.005 −5.369 10LEU13 N −28.901 18.567 −6.21 11 LEU13 CA −30.014 17.612 −6.169 12 LEU13C −29.844 16.617 −7.329 13 LEU13 O −29.62 15.424 −7.12 14 LEU13 CB−31.372 18.325 −6.222 15 LEU13 CG −31.708 19.142 −4.957 16 LEU13 CD1−33.003 19.928 −5.179 17 LEU13 CD2 −31.852 18.264 −3.713 18 ALA14 N−29.896 17.156 −8.611 19 ALA14 CA −30.062 16.233 −9.738 20 ALA14 C−28.974 15.148 −9.82 21 ALA14 O −29.304 13.976 −10.052 22 ALA14 CB−30.199 16.966 −11.065 23 PRO15 N −27.648 15.468 −9.615 24 PRO15 CA−26.614 14.433 −9.739 25 PRO15 C −26.577 13.384 −8.613 26 PRO15 O−25.683 12.539 −8.564 27 PRO15 CB −25.291 15.205 −9.757 28 PRO15 CG−25.689 16.611 −10.179 29 PRO15 CD −27.049 16.79 −9.529 30 ARG16 N−27.611 13.432 −7.698 31 ARG16 CA −27.889 12.313 −6.808 32 ARG16 C−28.719 11.222 −7.523 33 ARG16 O −28.811 10.092 −7.04 34 ARG16 CB−28.669 12.759 −5.565 35 ARG16 CG −27.998 13.889 −4.779 36 ARG16 CD−28.841 14.271 −3.563 37 ARG16 NE −28.553 15.641 −3.125 38 ARG16 CZ−28.879 16.152 −1.919 39 ARG16 NH1 −28.577 17.443 −1.675 40 ARG16 NH2−29.498 15.43 −0.966 41 TYR17 N −29.447 11.628 −8.629 42 TYR17 CA−30.387 10.734 −9.307 43 TYR17 C −30.365 10.753 −10.847 44 TYR17 O−30.892 9.844 −11.493 45 TYR17 CB −31.82 10.861 −8.763 46 TYR17 CG−32.42 12.247 −8.83 47 TYR17 CD1 −33.026 12.713 −10.005 48 TYR17 CD2−32.399 13.079 −7.7 49 TYR17 CE1 −33.605 13.985 −10.05 50 TYR17 CE2−32.976 14.348 −7.743 51 TYR17 CZ −33.575 14.791 −8.915 52 TYR17 OH−34.113 16.045 −8.919 53 SER18 N −29.786 11.842 −11.463 54 SER18 CA−29.362 11.756 −12.86 55 SER18 C −28.037 10.969 −12.906 56 SER18 O−27.418 10.622 −11.898 57 SER18 CB −29.251 13.135 −13.513 58 SER18 OG−28.18 13.894 −12.961 59 ARG19 N −27.608 10.612 −14.178 60 ARG19 CA−26.407 9.799 −14.302 61 ARG19 C −25.186 10.715 −14.173 62 ARG19 O−25.093 11.798 −14.749 63 ARG19 CB −26.36 9.052 −15.639 64 ARG19 CG−27.385 7.915 −15.684 65 ARG19 CD −27.352 7.176 −17.017 66 ARG19 NE−28.229 5.993 −17.009 67 ARG19 CZ −29.584 6.014 −17.06 68 ARG19 NH1−30.264 4.849 −17.011 69 ARG19 NH2 −30.28 7.164 −17.165 70 ARG20 N−24.137 10.172 −13.44 71 ARG20 CA −23.074 11.059 −12.946 72 ARG20 C−22.105 11.601 −14.025 73 ARG20 O −21.196 12.384 −13.749 74 ARG20 CB−22.298 10.391 −11.796 75 ARG20 CG −21.451 9.183 −12.222 76 ARG20 CD−21.741 7.92 −11.41 77 ARG20 NE −21.225 7.976 −10.034 78 ARG20 CZ−21.316 6.918 −9.177 79 ARG20 NH1 −20.707 6.953 −7.974 80 ARG20 NH2−22.006 5.801 −9.504 81 ALA21 N −22.342 11.167 −15.311 82 ALA21 CA−21.702 11.786 −16.466 83 ALA21 C −22.401 13.094 −16.884 84 ALA21 O−21.802 13.973 −17.513 85 ALA21 CB −21.7 10.815 −17.638 86 SER22 N−23.748 13.189 −16.596 87 SER22 CA −24.595 14.209 −17.204 88 SER22 C−24.187 15.655 −16.886 89 SER22 O −24.338 16.517 −17.76 90 SER22 CB−26.076 14.009 −16.861 91 SER22 OG −26.564 12.755 −17.346 92 PRO23 N−23.727 15.993 −15.627 93 PRO23 CA −23.271 17.363 −15.354 94 PRO23 C−21.806 17.591 −15.778 95 PRO23 O −21.311 18.716 −15.797 96 PRO23 CB−23.413 17.512 −13.839 97 PRO23 CG −23.201 16.095 −13.327 98 PRO23 CD−23.881 15.239 −14.385 99 GLN24 N −21.082 16.438 −16.016 100 GLN24 CA−19.678 16.471 −16.414 101 GLN24 C −19.544 16.633 −17.939 102 GLN24 O−18.62 17.275 −18.441 103 GLN24 CB −18.95 15.209 −15.942 104 GLN24 CG−18.96 15.085 −14.417 105 GLN24 CD −18.096 13.933 −13.964 106 GLN24 OE1−16.973 14.097 −13.504 107 GLN24 NE2 −18.651 12.698 −14.155 108 GLN25 N−20.485 15.967 −18.706 109 GLN25 CA −20.388 15.96 −20.167 110 GLN25 C−20.253 17.391 −20.748 111 GLN25 O −19.364 17.623 −21.581 112 GLN25 CB−21.577 15.23 −20.803 113 GLN25 CG −21.36 13.719 −20.86 114 GLN25 CD−22.653 13.001 −21.172 115 GLN25 OE1 −23.272 12.342 −20.342 116 GLN25NE2 −23.097 13.155 −22.455 117 PRO26 N −21.115 18.387 −20.32 118 PRO26CA −20.991 19.766 −20.799 119 PRO26 C −19.946 20.596 −20.02 120 PRO26 O−20.004 21.825 −19.936 121 PRO26 CB −22.395 20.35 −20.632 122 PRO26 CG−22.91 19.622 −19.398 123 PRO26 CD −22.368 18.214 −19.591 124 GLN27 N−18.849 19.895 −19.569 125 GLN27 CA −17.613 20.528 −19.115 126 GLN27 C−16.463 19.898 −19.929 127 GLN27 O −15.331 19.73 −19.484 128 GLN27 CB−17.416 20.396 −17.603 129 GLN27 CG −18.523 21.051 −16.774 130 GLN27 CD−18.442 22.561 −16.807 131 GLN27 OE1 −17.735 23.205 −16.038 132 GLN27NE2 −19.197 23.17 −17.767 133 GLN28 N −16.808 19.71 −21.262 134 GLN28 CA−15.945 19.054 −22.232 135 GLN28 C −16.691 18.924 −23.579 136 GLN28 O−16.447 19.642 −24.544 137 GLN28 CB −14.567 19.723 −22.419 138 GLN28 CG−14.595 21.247 −22.555 139 GLN28 CD −13.26 21.765 −23.043 140 GLN28 OE1−13.065 22.155 −24.189 141 GLN28 NE2 −12.242 21.746 −22.12 142 ASP29 N−17.633 17.907 −23.602 143 ASP29 CA −18.243 17.418 −24.852 144 ASP29 C−17.114 16.809 −25.738 145 ASP29 O −15.94 16.761 −25.373 146 ASP29 CB−19.095 18.476 −25.541 147 ASP29 CG −20.157 17.743 −26.352 148 ASP29 OD1−21.32 17.755 −25.873 149 ASP29 OD2 −19.723 17.189 −27.416 150 PHE30 N−17.515 16.262 −26.934 151 PHE30 CA −16.585 15.682 −27.911 152 PHE30 C−17.336 15.656 −29.244 153 PHE30 O −16.883 16.173 −30.268 154 PHE30 CB−16.091 14.297 −27.475 155 PHE30 CG −15.177 13.595 −28.452 156 PHE30 CD1−13.793 13.795 −28.41 157 PHE30 CD2 −15.69 12.688 −29.387 158 PHE30 CE1−12.94 13.077 −29.25 159 PHE30 CE2 −14.838 11.962 −30.221 160 PHE30 CZ−13.461 12.156 −30.153 161 GLU31 N −18.571 15.047 −29.206 162 GLU31 CA−19.371 14.861 −30.403 163 GLU31 C −19.84 16.233 −30.919 164 GLU31 O−19.914 16.467 −32.129 165 GLU31 CB −20.581 13.945 −30.146 166 GLU31 CG−20.204 12.473 −29.906 167 GLU31 CD −19.422 12.179 −28.631 168 GLU31 OE1−18.905 11.018 −28.535 169 GLU31 OE2 −19.275 13.143 −27.826 170 ALA32 N−20.23 17.156 −29.959 171 ALA32 CA −20.695 18.481 −30.361 172 ALA32 C−19.529 19.381 −30.805 173 ALA32 O −19.719 20.395 −31.476 174 ALA32 CB−21.475 19.175 −29.256 175 LEU33 N −18.285 19.002 −30.336 176 LEU33 CA−17.064 19.644 −30.808 177 LEU33 C −16.734 19.113 −32.211 178 LEU33 O−16.436 19.883 −33.13 179 LEU33 CB −15.891 19.427 −29.85 180 LEU33 CG−16.068 20.121 −28.484 181 LEU33 CD1 −14.97 19.656 −27.528 182 LEU33 CD2−16.034 21.645 −28.603 183 LEU34 N −16.806 17.743 −32.408 184 LEU34 CA−16.484 17.2 −33.728 185 LEU34 C −17.468 17.783 −34.767 186 LEU34 O−17.124 18.027 −35.925 187 LEU34 CB −16.582 15.672 −33.798 188 LEU34 CG−15.405 14.894 −33.178 189 LEU34 CD1 −15.661 13.393 −33.352 190 LEU34CD2 −14.056 15.246 −33.805 191 ALA35 N −18.765 17.953 −34.31 192 ALA35CA −19.818 18.481 −35.163 193 ALA35 C −19.724 20.006 −35.38 194 ALA35 O−20.487 20.59 −36.158 195 ALA35 CB −21.195 18.145 −34.609 196 GLU36 N−18.78 20.682 −34.645 197 GLU36 CA −18.303 22.015 −35.021 198 GLU36 C−17.186 21.785 −36.053 199 GLU36 O −17.234 22.262 −37.192 200 GLU36 CB−17.861 22.801 −33.778 201 GLU36 CG −17.443 24.24 −34.073 202 GLU36 CD−16.03 24.44 −34.627 203 GLU36 OE1 −15.172 23.573 −34.311 204 GLU36 OE2−15.902 25.463 −35.368 205 CYS37 N −16.146 20.978 −35.621 206 CYS37 CA−14.854 21.039 −36.301 207 CYS37 C −14.982 20.521 −37.736 208 CYS37 O−14.408 21.084 −38.67 209 CYS37 CB −13.796 20.222 −35.56 210 CYS37 SG−13.318 20.986 −33.971 211 LEU38 N −15.724 19.365 −37.893 212 LEU38 CA−15.839 18.71 −39.193 213 LEU38 C −16.86 19.396 −40.122 214 LEU38 O−16.939 19.086 −41.311 215 LEU38 CB −16.189 17.219 −39.063 216 LEU38 CG−14.967 16.304 −38.849 217 LEU38 CD1 −14.173 16.629 −37.586 218 LEU38CD2 −15.421 14.844 −38.807 219 ARG39 N −17.699 20.323 −39.529 220 ARG39CA −18.489 21.215 −40.371 221 ARG39 C −17.655 22.435 −40.786 222 ARG39 O−17.723 22.893 −41.927 223 ARG39 CB −19.765 21.696 −39.67 224 ARG39 CG−20.879 20.645 −39.722 225 ARG39 CD −22.192 21.193 −39.164 226 ARG39 NE−22.073 21.461 −37.731 227 ARG39 CZ −22.814 22.311 −37 228 ARG39 NH1−22.502 22.479 −35.699 229 ARG39 NH2 −23.855 22.991 −37.519 230 ASN40 N−16.956 23.056 −39.765 231 ASN40 CA −16.365 24.376 −39.99 232 ASN40 C−15.001 24.28 −40.69 233 ASN40 O −14.541 25.23 −41.324 234 ASN40 CB−16.187 25.141 −38.682 235 ASN40 CG −17.481 25.764 −38.204 236 ASN40 OD1−18.559 25.676 −38.782 237 ASN40 ND2 −17.346 26.478 −37.045 238 GLY41 N−14.267 23.143 −40.414 239 GLY41 CA −12.89 23.005 −40.854 240 GLY41 C−11.898 23.611 −39.86 241 GLY41 O −10.739 23.874 −40.176 242 CYS42 N−12.38 23.728 −38.571 243 CYS42 CA −11.519 24.131 −37.462 244 CYS42 C−10.8 22.878 −36.938 245 CYS42 O −11.253 21.744 −37.079 246 CYS42 CB−12.322 24.733 −36.301 247 CYS42 SG −13.088 26.325 −36.732 248 LEU43 N−9.636 23.151 −36.236 249 LEU43 CA −9.096 22.166 −35.302 250 LEU43 C−9.601 22.594 −33.916 251 LEU43 O −9.9 23.761 −33.659 252 LEU43 CB−7.562 22.161 −35.29 253 LEU43 CG −6.909 21.736 −36.619 254 LEU43 CD1−5.39 21.902 −36.521 255 LEU43 CD2 −7.243 20.293 −36.997 256 PHE44 N−9.616 21.585 −32.979 257 PHE44 CA −10.012 21.847 −31.602 258 PHE44 C−8.796 22.437 −30.875 259 PHE44 O −7.718 21.842 −30.797 260 PHE44 CB−10.433 20.557 −30.896 261 PHE44 CG −10.682 20.72 −29.416 262 PHE44 CD1−11.728 21.521 −28.942 263 PHE44 CD2 −9.854 20.066 −28.491 264 PHE44 CE1−11.942 21.656 −27.57 265 PHE44 CE2 −10.09 20.179 −27.123 266 PHE44 CZ−11.134 20.973 −26.663 267 GLU45 N −9.013 23.684 −30.325 268 GLU45 CA−8.117 24.256 −29.33 269 GLU45 C −8.867 24.183 −27.994 270 GLU45 O−10.022 24.587 −27.87 271 GLU45 CB −7.785 25.72 −29.653 272 GLU45 CG−6.759 25.864 −30.78 273 GLU45 CD −5.434 25.234 −30.385 274 GLU45 OE1−4.99 25.529 −29.227 275 GLU45 OE2 −4.95 24.381 −31.195 276 ASP46 N−8.134 23.636 −26.957 277 ASP46 CA −8.68 23.586 −25.607 278 ASP46 C−8.389 24.957 −24.968 279 ASP46 O −7.252 25.424 −24.869 280 ASP46 CB−8.053 22.452 −24.808 281 ASP46 CG −8.813 22.241 −23.517 282 ASP46 OD1−8.966 23.266 −22.785 283 ASP46 OD2 −9.206 21.058 −23.267 284 THR47 N−9.545 25.589 −24.549 285 THR47 CA −9.557 26.883 −23.87 286 THR47 C−9.369 26.703 −22.354 287 THR47 O −9.032 27.646 −21.638 288 THR47 CB−10.923 27.57 −24.096 289 THR47 OG1 −11.445 27.165 −25.369 290 THR47 CG2−10.813 29.09 −24.071 291 SER48 N −9.771 25.476 −21.842 292 SER48 CA−9.616 25.195 −20.406 293 SER48 C −8.158 24.878 −20.053 294 SER48 O−7.708 25.08 −18.927 295 SER48 CB −10.481 23.997 −20.005 296 SER48 OG−11.808 24.115 −20.522 297 PHE49 N −7.444 24.246 −21.05 298 PHE49 CA−6.033 23.891 −20.932 299 PHE49 C −5.262 24.642 −22.045 300 PHE49 O−4.968 24.108 −23.124 301 PHE49 CB −5.884 22.368 −21.029 302 PHE49 CG−4.649 21.869 −20.32 303 PHE49 CD1 −4.611 21.823 −18.919 304 PHE49 CD2−3.528 21.454 −21.044 305 PHE49 CE1 −3.482 21.342 −18.255 306 PHE49 CE2−2.404 20.966 −20.375 307 PHE49 CZ −2.382 20.901 −18.983 308 PRO50 N−4.967 25.977 −21.797 309 PRO50 CA −4.379 26.826 −22.826 310 PRO50 C−2.846 26.69 −22.873 311 PRO50 O −2.174 26.135 −22.003 312 PRO50 CB−4.778 28.243 −22.411 313 PRO50 CG −4.787 28.153 −20.889 314 PRO50 CD−5.383 26.774 −20.646 315 ALA51 N −2.291 27.32 −23.972 316 ALA51 CA−0.862 27.308 −24.27 317 ALA51 C −0.137 28.512 −23.632 318 ALA51 O 0.62329.243 −24.266 319 ALA51 CB −0.667 27.281 −25.782 320 THR52 N −0.34328.652 −22.272 321 THR52 CA 0.396 29.632 −21.46 322 THR52 C 1.448 28.845−20.633 323 THR52 O 1.924 27.777 −21.027 324 THR52 CB −0.56 30.482−20.595 325 THR52 OG1 −1.056 29.691 −19.513 326 THR52 CG2 −1.734 31.065−21.37 327 LEU53 N 1.866 29.437 −19.452 328 LEU53 CA 2.574 28.681−18.418 329 LEU53 C 1.6 28.189 −17.317 330 LEU53 O 1.975 27.466 −16.389331 LEU53 CB 3.658 29.545 −17.754 332 LEU53 CG 4.727 30.104 −18.714 333LEU53 CD1 5.702 30.996 −17.939 334 LEU53 CD2 5.501 29 −19.432 335 SER54N 0.291 28.613 −17.427 336 SER54 CA −0.681 28.479 −16.33 337 SER54 C−1.097 27.015 −16.162 338 SER54 O −1.541 26.571 −15.109 339 SER54 CB−1.966 29.285 −16.585 340 SER54 OG −1.692 30.559 −17.187 341 SER55 N−1.042 26.303 −17.334 342 SER55 CA −1.355 24.887 −17.495 343 SER55 C−0.139 24.005 −17.146 344 SER55 O −0.226 22.783 −17.014 345 SER55 CB−1.784 24.668 −18.951 346 SER55 OG −1.052 25.563 −19.803 347 ILE56 N1.066 24.675 −17.053 348 ILE56 CA 2.278 24.001 −16.601 349 ILE56 C 2.3324.124 −15.072 350 ILE56 O 2.39 23.12 −14.359 351 ILE56 CB 3.557 24.544−17.287 352 ILE56 CG1 3.357 24.643 −18.815 353 ILE56 CG2 4.77 23.668−16.944 354 ILE56 CD1 4.592 25.09 −19.577 355 GLY57 N 2.367 25.424−14.596 356 GLY57 CA 2.7 25.698 −13.211 357 GLY57 C 4.2 25.489 −12.997358 GLY57 O 4.879 24.744 −13.702 359 SER58 N 4.733 26.171 −11.92 360SER58 CA 6.169 26.079 −11.675 361 SER58 C 6.538 26.776 −10.355 362 SER58O 6.339 27.974 −10.161 363 SER58 CB 6.996 26.674 −12.826 364 SER58 OG6.453 27.908 −13.297 365 GLY59 N 7.11 25.938 −9.41 366 GLY59 CA 7.48826.438 −8.1 367 GLY59 C 8.997 26.684 −7.979 368 GLY59 O 9.805 25.782−7.739 369 SER60 N 9.412 27.977 −8.223 370 SER60 CA 10.761 28.487 −7.965371 SER60 C 11.834 27.742 −8.76 372 SER60 O 12.293 28.182 −9.813 373SER60 CB 11.133 28.6 −6.484 374 SER60 OG 12.351 29.365 −6.324 375 LEU61N 12.211 26.514 −8.255 376 LEU61 CA 13.268 25.74 −8.903 377 LEU61 C12.877 25.429 −10.352 378 LEU61 O 13.704 25.396 −11.26 379 LEU61 CB13.547 24.434 −8.149 380 LEU61 CG 14.084 24.624 −6.716 381 LEU61 CD114.196 23.264 −6.022 382 LEU61 CD2 15.443 25.326 −6.691 383 LEU62 N11.543 25.141 −10.533 384 LEU62 CA 11.016 24.696 −11.813 385 LEU62 C10.583 25.875 −12.708 386 LEU62 O 10.123 25.694 −13.835 387 LEU62 CB9.873 23.701 −11.583 388 LEU62 CG 10.292 22.481 −10.728 389 LEU62 CD19.07 21.645 −10.371 390 LEU62 CD2 11.333 21.609 −11.429 391 GLN63 N10.796 27.141 −12.19 392 GLN63 CA 10.621 28.352 −12.999 393 GLN63 C11.905 28.667 −13.786 394 GLN63 O 11.905 29.443 −14.74 395 GLN63 CB10.28 29.576 −12.132 396 GLN63 CG 8.901 29.468 −11.493 397 GLN63 CD8.643 30.538 −10.459 398 GLN63 OE1 8.52 30.29 −9.262 399 GLN63 NE2 8.57431.811 −10.947 400 LYS64 N 13.058 28.134 −13.247 401 LYS64 CA 14.39428.557 −13.668 402 LYS64 C 14.973 27.615 −14.741 403 LYS64 O 15.96427.927 −15.399 404 LYS64 CB 15.316 28.621 −12.437 405 LYS64 CG 14.86329.702 −11.435 406 LYS64 CD 15.239 29.355 −9.991 407 LYS64 CE 14.47130.217 −8.988 408 LYS64 NZ 14.487 29.568 −7.652 409 LEU65 N 14.36626.377 −14.827 410 LEU65 CA 14.828 25.348 −15.748 411 LEU65 C 14.29925.454 −17.198 412 LEU65 O 15.052 25.083 −18.109 413 LEU65 CB 14.56223.925 −15.22 414 LEU65 CG 15.32 23.551 −13.931 415 LEU65 CD1 14.88522.157 −13.472 416 LEU65 CD2 16.838 23.581 −14.107 417 PRO66 N 12.98525.815 −17.464 418 PRO66 CA 12.437 25.65 −18.822 419 PRO66 C 12.08726.965 −19.575 420 PRO66 O 10.97 27.132 −20.083 421 PRO66 CB 11.16724.841 −18.536 422 PRO66 CG 10.629 25.517 −17.279 423 PRO66 CD 11.89325.854 −16.493 424 PRO67 N 13.064 27.918 −19.775 425 PRO67 CA 12.78229.095 −20.588 426 PRO67 C 12.822 28.72 −22.081 427 PRO67 O 13.39127.717 −22.509 428 PRO67 CB 13.909 30.063 −20.243 429 PRO67 CG 15.08529.13 −19.974 430 PRO67 CD 14.441 27.918 −19.309 431 ARG68 N 12.17929.621 −22.908 432 ARG68 CA 12.145 29.51 −24.371 433 ARG68 C 11.21728.398 −24.898 434 ARG68 O 11.203 28.074 −26.089 435 ARG68 CB 13.52529.367 −25.038 436 ARG68 CG 14.512 30.474 −24.677 437 ARG68 CD 15.85230.291 −25.393 438 ARG68 NE 16.958 30.148 −24.436 439 ARG68 CZ 17.25229.02 −23.743 440 ARG68 NH1 18.155 29.105 −22.735 441 ARG68 NH2 16.70127.826 −23.987 442 LEU69 N 10.338 27.861 −23.97 443 LEU69 CA 9.58426.677 −24.351 444 LEU69 C 8.561 27.022 −25.444 445 LEU69 O 8.092 28.147−25.609 446 LEU69 CB 8.961 25.93 −23.163 447 LEU69 CG 8.04 26.719−22.211 448 LEU69 CD1 6.717 27.144 −22.848 449 LEU69 CD2 7.734 25.845−20.99 450 GLN70 N 8.218 25.944 −26.234 451 GLN70 CA 7.169 26.042−27.225 452 GLN70 C 6.106 25.004 −26.851 453 GLN70 O 6.376 23.939−26.294 454 GLN70 CB 7.673 25.793 −28.652 455 GLN70 CG 8.902 26.625−29.028 456 GLN70 CD 10.18 25.816 −28.919 457 GLN70 OE1 10.366 24.784−29.564 458 GLN70 NE2 11.135 26.334 −28.095 459 TRP71 N 4.834 25.353−27.245 460 TRP71 CA 3.735 24.391 −27.223 461 TRP71 C 3.541 24.022−28.697 462 TRP71 O 3.381 24.894 −29.553 463 TRP71 CB 2.456 25.02 −26.68464 TRP71 CG 2.454 25.224 −25.191 465 TRP71 CD1 2.869 26.355 −24.508 466TRP71 CD2 1.907 24.336 −24.212 467 TRP71 NE1 2.537 26.21 −23.186 468TRP71 CE2 1.903 25.006 −22.992 469 TRP71 CE3 1.334 23.052 −24.262 470TRP71 CZ2 1.291 24.491 −21.846 471 TRP71 CZ3 0.686 22.535 −23.136 472TRP71 CH2 0.656 23.252 −21.947 473 LYS72 N 3.624 22.679 −28.983 474LYS72 CA 3.494 22.16 −30.348 475 LYS72 C 2.42 21.068 −30.347 476 LYS72 O2.092 20.469 −29.321 477 LYS72 CB 4.83 21.592 −30.849 478 LYS72 CG 5.85822.692 −31.13 479 LYS72 CD 7.126 22.114 −31.759 480 LYS72 CE 8.19123.186 −31.926 481 LYS72 NZ 9.428 22.568 −32.457 482 ARG73 N 1.87120.801 −31.584 483 ARG73 CA 1.094 19.583 −31.812 484 ARG73 C 2.11518.449 −32.15 485 ARG73 O 3.335 18.648 −32.191 486 ARG73 CB 0.057 19.819−32.924 487 ARG73 CG −1 20.889 −32.607 488 ARG73 CD −2.153 20.364−31.751 489 ARG73 NE −3.204 21.384 −31.608 490 ARG73 CZ −4.491 21.125−31.262 491 ARG73 NH1 −4.929 19.909 −30.901 492 ARG73 NH2 −5.372 22.129−31.273 493 PRO74 N 1.613 17.176 −32.386 494 PRO74 CA 2.532 16.075−32.691 495 PRO74 C 3.204 16.037 −34.08 496 PRO74 O 4.339 15.537 −34.178497 PRO74 CB 1.692 14.815 −32.46 498 PRO74 CG 0.722 15.264 −31.377 499PRO74 CD 0.364 16.664 −31.838 500 PRO75 N 2.518 16.432 −35.214 501 PRO75CA 3.119 16.245 −36.536 502 PRO75 C 4.309 17.176 −36.837 503 PRO75 O5.012 17.021 −37.836 504 PRO75 CB 1.974 16.435 −37.528 505 PRO75 CG1.003 17.338 −36.786 506 PRO75 CD 1.147 16.902 −35.335 507 GLU76 N 4.5518.16 −35.895 508 GLU76 CA 5.745 18.993 −35.974 509 GLU76 C 6.967 18.157−35.531 510 GLU76 O 8.116 18.48 −35.833 511 GLU76 CB 5.704 20.206−35.021 512 GLU76 CG 4.624 21.241 −35.323 513 GLU76 CD 3.326 20.806−34.659 514 GLU76 OE1 2.924 21.524 −33.694 515 GLU76 OE2 2.825 19.744−35.135 516 LEU77 N 6.674 17.174 −34.601 517 LEU77 CA 7.698 16.407−33.913 518 LEU77 C 7.993 15.098 −34.656 519 LEU77 O 9.151 14.751−34.894 520 LEU77 CB 7.281 16.104 −32.465 521 LEU77 CG 7.016 17.359−31.608 522 LEU77 CD1 6.472 16.945 −30.239 523 LEU77 CD2 8.274 18.211−31.436 524 HIS78 N 6.902 14.283 −34.897 525 HIS78 CA 7.056 12.996−35.577 526 HIS78 C 6.321 13.054 −36.919 527 HIS78 O 5.266 13.665−37.072 528 HIS78 CB 6.494 11.831 −34.748 529 HIS78 CG 7.297 11.485−33.533 530 HIS78 ND1 6.849 10.582 −32.599 531 HIS78 CD2 8.531 11.874−33.051 532 HIS78 CE1 7.78 10.503 −31.605 533 HIS78 NE2 8.817 11.265−31.855 534 SER79 N 6.896 12.292 −37.918 535 SER79 CA 6.39 12.314−39.296 536 SER79 C 5.22 11.341 −39.492 537 SER79 O 4.445 11.438 −40.442538 SER79 CB 7.51 11.959 −40.277 539 SER79 OG 8.239 10.828 −39.793 540ASN80 N 5.183 10.301 −38.59 541 ASN80 CA 4.117 9.304 −38.557 542 ASN80 C3.642 9.227 −37.097 543 ASN80 O 4.05 8.344 −36.336 544 ASN80 CB 4.6527.957 −39.044 545 ASN80 CG 3.555 6.92 −39.15 546 ASN80 OD1 2.364 7.137−38.952 547 ASN80 ND2 4.004 5.678 −39.506 548 PRO81 N 2.793 10.222−36.654 549 PRO81 CA 2.231 10.173 −35.311 550 PRO81 C 1.077 9.157 −35.31551 PRO81 O 0.264 9.064 −36.229 552 PRO81 CB 1.737 11.592 −35.063 553PRO81 CG 1.329 12.074 −36.452 554 PRO81 CD 2.346 11.41 −37.374 555 GLN82N 1.042 8.343 −34.196 556 GLN82 CA 0.079 7.257 −34.071 557 GLN82 C−0.393 7.201 −32.612 558 GLN82 O 0.262 7.684 −31.692 559 GLN82 CB 0.6875.91 −34.478 560 GLN82 CG 1.026 5.81 −35.967 561 GLN82 CD −0.208 5.864−36.841 562 GLN82 OE1 −1.346 5.673 −36.422 563 GLN82 NE2 0.056 6.091−38.163 564 PHE83 N −1.617 6.574 −32.457 565 PHE83 CA −2.289 6.467−31.154 566 PHE83 C −2.105 5.038 −30.619 567 PHE83 O −1.725 4.817−29.472 568 PHE83 CB −3.772 6.838 −31.314 569 PHE83 CG −4.503 7.095−30.019 570 PHE83 CD1 −4.162 8.191 −29.213 571 PHE83 CD2 −5.58 6.284−29.64 572 PHE83 CE1 −4.89 8.472 −28.055 573 PHE83 CE2 −6.306 6.572−28.483 574 PHE83 CZ −5.965 7.666 −27.693 575 TYR84 N −2.472 4.037−31.493 576 TYR84 CA −2.292 2.612 −31.215 577 TYR84 C −2.348 1.943−32.592 578 TYR84 O −3.064 2.387 −33.49 579 TYR84 CB −3.419 2.024−30.349 580 TYR84 CG −3.01 1.729 −28.926 581 TYR84 CD1 −3.451 2.547−27.877 582 TYR84 CD2 −2.2 0.621 −28.633 583 TYR84 CE1 −3.109 2.253−26.556 584 TYR84 CE2 −1.846 0.336 −27.314 585 TYR84 CZ −2.305 1.151−26.286 586 TYR84 OH −1.942 0.839 −25.008 587 PHE85 N −1.583 0.803−32.722 588 PHE85 CA −1.557 0.054 −33.976 589 PHE85 C −1.018 −1.353−33.675 590 PHE85 O 0.157 −1.675 −33.809 591 PHE85 CB −0.869 0.752−35.162 592 PHE85 CG 0.567 1.2 −35.04 593 PHE85 CD1 1.526 0.717 −35.943594 PHE85 CD2 0.952 2.184 −34.122 595 PHE85 CE1 2.828 1.22 −35.939 596PHE85 CE2 2.257 2.675 −34.109 597 PHE85 CZ 3.192 2.205 −35.026 598 ALA86N −2.007 −2.183 −33.163 599 ALA86 CA −1.777 −3.512 −32.605 600 ALA86 C−1.378 −3.41 −31.12 601 ALA86 O −1.093 −2.352 −30.564 602 ALA86 CB−0.813 −4.383 −33.405 603 LYS87 N −1.392 −4.628 −30.458 604 LYS87 CA−1.132 −4.726 −29.017 605 LYS87 C 0.379 −4.824 −28.732 606 LYS87 O 0.853−4.568 −27.626 607 LYS87 CB −1.863 −5.937 −28.42 608 LYS87 CG −3.355−5.643 −28.213 609 LYS87 CD −4.1 −6.829 −27.594 610 LYS87 CE −5.384−6.375 −26.912 611 LYS87 NZ −6.065 −7.531 −26.331 612 ALA88 N 1.123−5.333 −29.781 613 ALA88 CA 2.584 −5.318 −29.775 614 ALA88 C 3.128−6.294 −28.7 615 ALA88 O 2.602 −7.389 −28.494 616 ALA88 CB 3.115 −3.89−29.745 617 LYS89 N 4.291 −5.911 −28.057 618 LYS89 CA 4.945 −6.782−27.079 619 LYS89 C 5.754 −5.89 −26.126 620 LYS89 O 5.385 −5.663 −24.971621 LYS89 CB 5.825 −7.83 −27.788 622 LYS89 CG 6.606 −8.738 −26.825 623LYS89 CD 7.485 −9.72 −27.607 624 LYS89 CE 8.029 −10.844 −26.742 625LYS89 NZ 9.338 −10.527 −26.142 626 ARG90 N 6.881 −5.351 −26.721 627ARG90 CA 7.917 −4.637 −25.989 628 ARG90 C 7.456 −3.225 −25.613 629 ARG90O 6.494 −2.665 −26.128 630 ARG90 CB 9.216 −4.573 −26.818 631 ARG90 CG10.046 −5.857 −26.712 632 ARG90 CD 10.86 −5.887 −25.426 633 ARG90 NE11.405 −7.218 −25.152 634 ARG90 CZ 12.701 −7.569 −25.276 635 ARG90 NH113.526 −6.914 −26.122 636 ARG90 NH2 13.203 −8.576 −24.542 637 LEU91 N8.235 −2.676 −24.617 638 LEU91 CA 7.904 −1.434 −23.919 639 LEU91 C 9.226−0.664 −23.931 640 LEU91 O 10.235 −1.123 −23.392 641 LEU91 CB 7.475−1.815 −22.498 642 LEU91 CG 6.487 −0.843 −21.846 643 LEU91 CD1 5.945−1.465 −20.56 644 LEU91 CD2 7.114 0.509 −21.527 645 ASP92 N 9.214 0.48−24.704 646 ASP92 CA 10.404 1.319 −24.881 647 ASP92 C 11.496 0.478−25.612 648 ASP92 O 11.245 −0.57 −26.213 649 ASP92 CB 10.877 1.95−23.572 650 ASP92 CG 9.809 2.796 −22.89 651 ASP92 OD1 9.046 3.441−23.668 652 ASP92 OD2 9.837 2.781 −21.626 653 LEU93 N 12.768 1.028−25.618 654 LEU93 CA 13.922 0.28 −26.105 655 LEU93 C 14.741 −0.175−24.876 656 LEU93 O 14.723 −1.344 −24.474 657 LEU93 CB 14.753 1.108−27.099 658 LEU93 CG 14.023 1.446 −28.416 659 LEU93 CD1 14.893 2.381−29.261 660 LEU93 CD2 13.669 0.2 −29.23 661 CYS94 N 15.447 0.831 −24.233662 CYS94 CA 16.324 0.577 −23.09 663 CYS94 C 16.131 1.7 −22.062 664CYS94 O 15.357 1.583 −21.113 665 CYS94 CB 17.803 0.423 −23.481 666 CYS94SG 18.154 −1.146 −24.337 667 GLN95 N 16.809 2.875 −22.309 668 GLN95 CA16.828 3.977 −21.343 669 GLN95 C 15.595 4.88 −21.519 670 GLN95 O 15.6666.101 −21.667 671 GLN95 CB 18.128 4.782 −21.468 672 GLN95 CG 19.35 3.971−21.037 673 GLN95 CD 20.631 4.716 −21.332 674 GLN95 OE1 21.398 4.387−22.23 675 GLN95 NE2 20.868 5.796 −20.532 676 GLY96 N 14.387 4.241−21.336 677 GLY96 CA 13.113 4.941 −21.412 678 GLY96 C 12.794 5.775−20.17 679 GLY96 O 11.648 5.904 −19.733 680 ILE97 N 13.836 6.527 −19.667681 ILE97 CA 13.733 7.329 −18.442 682 ILE97 C 13.041 8.673 −18.791 683ILE97 O 13.56 9.771 −18.609 684 ILE97 CB 15.122 7.531 −17.772 685 ILE97CG1 15.926 6.211 −17.692 686 ILE97 CG2 14.949 8.124 −16.362 687 ILE97CD1 17.34 6.378 −17.148 688 VAL98 N 11.751 8.517 −19.265 689 VAL98 CA10.927 9.621 −19.757 690 VAL98 C 9.429 9.286 −19.558 691 VAL98 O 8.5319.843 −20.19 692 VAL98 CB 11.221 9.994 −21.234 693 VAL98 CG1 12.47510.859 −21.382 694 VAL98 CG2 11.335 8.767 −22.145 695 GLY99 N 9.1858.415 −18.514 696 GLY99 CA 7.875 7.865 −18.235 697 GLY99 C 7.657 7.649−16.74 698 GLY99 O 8.578 7.612 −15.928 699 ASP100 N 6.324 7.452 −16.42700 ASP100 CA 5.893 7.06 −15.077 701 ASP100 C 5.918 5.515 −15.027 702ASP100 O 5.893 4.805 −16.031 703 ASP100 CB 4.487 7.593 −14.83 704 ASP100CG 4.12 7.565 −13.351 705 ASP100 OD1 3.671 8.643 −12.877 706 ASP100 OD24.264 6.429 −12.793 707 CYS101 N 5.865 4.992 −13.749 708 CYS101 CA 5.7693.556 −13.534 709 CYS101 C 4.373 3.044 −13.937 710 CYS101 O 4.226 1.887−14.348 711 CYS101 CB 6.133 3.221 −12.089 712 CYS101 SG 6.148 1.438−11.724 713 TRP102 N 3.29 3.904 −13.831 714 TRP102 CA 1.957 3.399−14.203 715 TRP102 C 1.878 3.08 −15.714 716 TRP102 O 1.076 2.263 −16.17717 TRP102 CB 0.751 4.239 −13.763 718 TRP102 CG 0.781 5.685 −14.133 719TRP102 CD1 1.058 6.715 −13.259 720 TRP102 CD2 0.498 6.294 −15.401 721TRP102 NE1 1.069 7.893 −13.955 722 TRP102 CE2 0.713 7.668 −15.262 723TRP102 CE3 0.088 5.808 −16.661 724 TRP102 CZ2 0.584 8.568 −16.325 725TRP102 CZ3 −0.042 6.694 −17.735 726 TRP102 CH2 0.205 8.052 −17.567 727PHE103 N 2.748 3.808 −16.5 728 PHE103 CA 2.834 3.674 −17.967 729 PHE103C 3.477 2.303 −18.263 730 PHE103 O 3.184 1.626 −19.246 731 PHE103 CB3.697 4.823 −18.521 732 PHE103 CG 3.647 5.182 −19.989 733 PHE103 CD12.98 4.439 −20.966 734 PHE103 CD2 4.345 6.338 −20.391 735 PHE103 CE13.011 4.843 −22.306 736 PHE103 CE2 4.371 6.743 −21.725 737 PHE103 CZ3.701 5.994 −22.684 738 LEU104 N 4.471 1.954 −17.371 739 LEU104 CA 5.1530.669 −17.416 740 LEU104 C 4.232 −0.452 −16.89 741 LEU104 O 4.292 −1.604−17.331 742 LEU104 CB 6.453 0.729 −16.608 743 LEU104 CG 7.691 1.153−17.425 744 LEU104 CD1 7.519 2.468 −18.183 745 LEU104 CD2 8.893 1.266−16.486 746 ALA105 N 3.406 −0.101 −15.834 747 ALA105 CA 2.56 −1.101−15.193 748 ALA105 C 1.429 −1.517 −16.151 749 ALA105 O 1.062 −2.692−16.237 750 ALA105 CB 1.951 −0.585 −13.898 751 ALA106 N 0.819 −0.484−16.85 752 ALA106 CA −0.432 −0.724 −17.57 753 ALA106 C −0.204 −1.675−18.754 754 ALA106 O −1.054 −2.479 −19.141 755 ALA106 CB −1.013 0.582−18.097 756 LEU107 N 1.009 −1.53 −19.398 757 LEU107 CA 1.321 −2.176−20.673 758 LEU107 C 1.718 −3.678 −20.575 759 LEU107 O 2.258 −4.293−21.501 760 LEU107 CB 2.384 −1.374 −21.433 761 LEU107 CG 1.878 −0.061−22.059 762 LEU107 CD1 3.035 0.663 −22.753 763 LEU107 CD2 0.74 −0.303−23.051 764 GLN108 N 1.244 −4.303 −19.443 765 GLN108 CA 1.063 −5.742−19.321 766 GLN108 C −0.337 −6.131 −19.827 767 GLN108 O −0.557 −7.222−20.355 768 GLN108 CB 1.183 −6.173 −17.853 769 GLN108 CG 2.621 −6.422−17.41 770 GLN108 CD 3.582 −5.274 −17.628 771 GLN108 OE1 4.64 −5.424−18.24 772 GLN108 NE2 3.248 −4.089 −17.05 773 ALA109 N −1.359 −5.255−19.525 774 ALA109 CA −2.772 −5.614 −19.668 775 ALA109 C −3.291 −5.495−21.117 776 ALA109 O −4.39 −5.022 −21.399 777 ALA109 CB −3.596 −4.765−18.713 778 LEU110 N −2.462 −6.089 −22.051 779 LEU110 CA −2.756 −6.142−23.476 780 LEU110 C −2.279 −7.502 −24.017 781 LEU110 O −1.669 −7.63−25.075 782 LEU110 CB −2.121 −5.001 −24.287 783 LEU110 CG −2.629 −3.591−23.935 784 LEU110 CD1 −1.731 −2.934 −22.892 785 LEU110 CD2 −2.658 −2.71−25.186 786 ALA111 N −2.72 −8.579 −23.265 787 ALA111 CA −2.753 −9.915−23.857 788 ALA111 C −4.101 −10.024 −24.608 789 ALA111 O −4.773 −9.031−24.91 790 ALA111 CB −2.558 −10.955 −22.762 791 LEU112 N −4.465 −11.29−25.015 792 LEU112 CA −5.86 −11.58 −25.354 793 LEU112 C −6.414 −12.161−24.046 794 LEU112 O −5.918 −13.188 −23.572 795 LEU112 CB −5.952 −12.64−26.461 796 LEU112 CG −5.261 −12.256 −27.785 797 LEU112 CD1 −5.34−13.427 −28.769 798 LEU112 CD2 −5.866 −11.005 −28.417 799 HIS113 N −7.43−11.444 −23.451 800 HIS113 CA −7.821 −11.611 −22.048 801 HIS113 C −6.923−10.665 −21.229 802 HIS113 O −5.785 −10.341 −21.566 803 HIS113 CB −7.778−13.012 −21.426 804 HIS113 CG −8.611 −14.02 −22.134 805 HIS113 ND1−8.095 −14.823 −23.122 806 HIS113 CD2 −9.929 −14.406 −22.032 807 HIS113CE1 −9.092 −15.645 −23.555 808 HIS113 NE2 −10.212 −15.424 −22.906 809GLN114 N −7.504 −10.22 −20.054 810 GLN114 CA −6.884 −9.215 −19.194 811GLN114 C −6.983 −7.797 −19.8 812 GLN114 O −6.316 −6.849 −19.385 813GLN114 CB −5.444 −9.521 −18.747 814 GLN114 CG −5.258 −10.965 −18.283 815GLN114 CD −4.009 −11.203 −17.462 816 GLN114 OE1 −4.009 −11.949 −16.481817 GLN114 NE2 −2.866 −10.614 −17.913 818 ASP115 N −7.986 −7.644 −20.739819 ASP115 CA −8.063 −6.457 −21.592 820 ASP115 C −8.661 −5.284 −20.776821 ASP115 O −9.86 −5.006 −20.77 822 ASP115 CB −8.938 −6.751 −22.812 823ASP115 CG −8.176 −7.678 −23.745 824 ASP115 OD1 −8.126 −8.901 −23.421 825ASP115 OD2 −7.654 −7.126 −24.764 826 ILE116 N −7.728 −4.588 −20.009 827ILE116 CA −8.165 −3.488 −19.133 828 ILE116 C −8.463 −2.254 −20.018 829ILE116 O −9.325 −1.423 −19.726 830 ILE116 CB −7.086 −3.125 −18.066 831ILE116 CG1 −6.977 −4.238 −16.998 832 ILE116 CG2 −7.362 −1.775 −17.38 833ILE116 CD1 −5.89 −3.988 −15.958 834 LEU117 N −7.566 −2.046 −21.058 835LEU117 CA −7.268 −0.678 −21.504 836 LEU117 C −8.491 0.028 −22.113 837LEU117 O −8.609 1.255 −22.1 838 LEU117 CB −6.122 −0.667 −22.53 839LEU117 CG −4.72 −0.396 −21.943 840 LEU117 CD1 −4.559 1.055 −21.489 841LEU117 CD2 −4.346 −1.348 −20.81 842 SER118 N −9.408 −0.805 −22.71 843SER118 CA −10.624 −0.306 −23.348 844 SER118 C −11.65 0.289 −22.366 845SER118 O −12.654 0.877 −22.767 846 SER118 CB −11.286 −1.413 −24.169 847SER118 OG −11.43 −2.584 −23.363 848 ARG119 N −11.355 0.143 −21.025 849ARG119 CA −12.143 0.802 −19.988 850 ARG119 C −11.707 2.275 −19.855 851ARG119 O −12.455 3.127 −19.381 852 ARG119 CB −11.894 0.161 −18.62 853ARG119 CG −12.335 −1.297 −18.522 854 ARG119 CD −11.739 −1.974 −17.293855 ARG119 NE −12.298 −1.457 −16.042 856 ARG119 CZ −11.803 −0.482−15.244 857 ARG119 NH1 −10.711 0.247 −15.555 858 ARG119 NH2 −12.452−0.258 −14.089 859 VAL120 N −10.362 2.488 −20.094 860 VAL120 CA −9.7063.779 −19.884 861 VAL120 C −9.694 4.555 −21.214 862 VAL120 O −9.9325.766 −21.253 863 VAL120 CB −8.269 3.569 −19.347 864 VAL120 CG1 −7.5294.896 −19.144 865 VAL120 CG2 −8.281 2.797 −18.023 866 VAL121 N −9.2423.824 −22.299 867 VAL121 CA −8.985 4.393 −23.615 868 VAL121 C −10.1373.956 −24.546 869 VAL121 O −10.169 2.831 −25.059 870 VAL121 CB −7.6293.889 −24.177 871 VAL121 CG1 −7.284 4.624 −25.476 872 VAL121 CG2 −6.4814.066 −23.177 873 PRO122 N −11.151 4.858 −24.793 874 PRO122 CA −12.2574.516 −25.681 875 PRO122 C −11.727 4.64 −27.122 876 PRO122 O −11.6555.707 −27.736 877 PRO122 CB −13.34 5.54 −25.351 878 PRO122 CG −12.566.749 −24.854 879 PRO122 CD −11.376 6.132 −24.132 880 LEU123 N −11.2653.444 −27.648 881 LEU123 CA −10.478 3.372 −28.894 882 LEU123 C −11.2853.658 −30.198 883 LEU123 O −11.001 3.142 −31.278 884 LEU123 CB −9.8021.984 −29.007 885 LEU123 CG −8.58 1.794 −28.085 886 LEU123 CD1 −8.2460.307 −27.948 887 LEU123 CD2 −7.356 2.544 −28.613 888 ASN124 N −12.2114.68 −30.117 889 ASN124 CA −12.829 5.29 −31.3 890 ASN124 C −12.104 6.607−31.619 891 ASN124 O −12.689 7.68 −31.747 892 ASN124 CB −14.341 5.477−31.162 893 ASN124 CG −14.79 6.026 −29.825 894 ASN124 OD1 −14.992 7.218−29.597 895 ASN124 ND2 −14.98 5.078 −28.858 896 GLN125 N −10.738 6.468−31.766 897 GLN125 CA −9.826 7.606 −31.832 898 GLN125 C −8.631 7.22−32.717 899 GLN125 O −8.277 6.051 −32.867 900 GLN125 CB −9.337 7.976−30.426 901 GLN125 CG −10.449 8.556 −29.557 902 GLN125 CD −9.954 8.892−28.177 903 GLN125 OE1 −9.565 10.014 −27.87 904 GLN125 NE2 −9.96 7.87−27.275 905 SER126 N −7.977 8.292 −33.292 906 SER126 CA −6.903 8.121−34.272 907 SER126 C −6.247 9.491 −34.529 908 SER126 O −6.72 10.537−34.089 909 SER126 CB −7.434 7.571 −35.601 910 SER126 OG −6.393 7.517−36.579 911 PHE127 N −5.107 9.413 −35.315 912 PHE127 CA −4.435 10.605−35.843 913 PHE127 C −4.861 10.96 −37.282 914 PHE127 O −4.327 11.876−37.907 915 PHE127 CB −2.908 10.456 −35.78 916 PHE127 CG −2.362 10.971−34.47 917 PHE127 CD1 −2.161 10.109 −33.388 918 PHE127 CD2 −2.091 12.334−34.312 919 PHE127 CE1 −1.653 10.589 −32.181 920 PHE127 CE2 −1.61212.819 −33.096 921 PHE127 CZ −1.378 11.946 −32.035 922 THR128 N −5.94510.264 −37.77 923 THR128 CA −6.502 10.531 −39.093 924 THR128 C −8.03410.544 −38.983 925 THR128 O −8.707 11.526 −39.309 926 THR128 CB −5.9929.555 −40.178 927 THR128 OG1 −5.571 8.303 −39.635 928 THR128 CG2 −4.78510.13 −40.915 929 GLU129 N −8.608 9.393 −38.494 930 GLU129 CA −10.0359.112 −38.601 931 GLU129 C −10.811 10.009 −37.614 932 GLU129 O −11.2059.625 −36.517 933 GLU129 CB −10.335 7.629 −38.31 934 GLU129 CG −9.7596.652 −39.339 935 GLU129 CD −8.271 6.311 −39.226 936 GLU129 OE1 −7.9295.198 −39.697 937 GLU129 OE2 −7.53 7.208 −38.687 938 LYS130 N −11.00511.296 −38.093 939 LYS130 CA −11.731 12.34 −37.373 940 LYS130 C −10.87712.95 −36.242 941 LYS130 O −11.355 13.334 −35.177 942 LYS130 CB −13.12611.929 −36.873 943 LYS130 CG −13.995 11.284 −37.956 944 LYS130 CD−15.417 11.04 −37.448 945 LYS130 CE −16.274 10.388 −38.522 946 LYS130 NZ−17.636 10.16 −37.984 947 TYR131 N −9.562 13.204 −36.609 948 TYR131 CA−8.563 13.568 −35.589 949 TYR131 C −8.9 14.925 −34.947 950 TYR131 O−8.836 15.109 −33.729 951 TYR131 CB −7.175 13.608 −36.26 952 TYR131 CG−6.046 14.306 −35.541 953 TYR131 CD1 −5.831 14.159 −34.166 954 TYR131CD2 −5.155 15.104 −36.281 955 TYR131 CE1 −4.787 14.844 −33.54 956 TYR131CE2 −4.101 15.77 −35.655 957 TYR131 CZ −3.927 15.641 −34.285 958 TYR131OH −2.881 16.305 −33.712 959 ALA132 N −9.049 15.977 −35.833 960 ALA132CA −9.562 17.291 −35.428 961 ALA132 C −8.797 18.069 −34.324 962 ALA132 O−9.201 19.152 −33.891 963 ALA132 CB −11.05 17.221 −35.103 964 GLY133 N−7.619 17.503 −33.892 965 GLY133 CA −6.856 18.065 −32.802 966 GLY133 C−7.431 17.783 −31.41 967 GLY133 O −7.144 18.534 −30.47 968 ILE134 N−8.174 16.624 −31.274 969 ILE134 CA −8.908 16.289 −30.044 970 ILE134 C−8.793 14.785 −29.724 971 ILE134 O −8.765 13.926 −30.603 972 ILE134 CB−10.397 16.728 −30.16 973 ILE134 CG1 −11.124 16.686 −28.801 974 ILE134CG2 −11.169 15.94 −31.224 975 ILE134 CD1 −12.511 17.313 −28.847 976PHE135 N −8.83 14.489 −28.371 977 PHE135 CA −8.833 13.117 −27.856 978PHE135 C −9.685 13.073 −26.568 979 PHE135 O −10.012 14.085 −25.944 980PHE135 CB −7.413 12.624 −27.534 981 PHE135 CG −6.553 12.377 −28.746 982PHE135 CD1 −5.529 13.267 −29.088 983 PHE135 CD2 −6.777 11.255 −29.554984 PHE135 CE1 −4.741 13.032 −30.215 985 PHE135 CE2 −6.003 11.033−30.691 986 PHE135 CZ −4.989 11.924 −31.023 987 ARG136 N −10.009 11.798−26.139 988 ARG136 CA −10.796 11.546 −24.941 989 ARG136 C −10.416 10.231−24.233 990 ARG136 O −9.984 9.248 −24.834 991 ARG136 CB −12.308 11.593−25.207 992 ARG136 CG −12.821 10.558 −26.217 993 ARG136 CD −14.35 10.528−26.238 994 ARG136 NE −14.865 9.659 −27.303 995 ARG136 CZ −16.136 9.742−27.779 996 ARG136 NH1 −16.503 8.988 −28.829 997 ARG136 NH2 −17.05410.552 −27.241 998 PHE137 N −10.687 10.254 −22.871 999 PHE137 CA −10.3219.168 −21.959 1000 PHE137 C −11.354 9.108 −20.824 1001 PHE137 O −11.94710.111 −20.421 1002 PHE137 CB −8.944 9.385 −21.316 1003 PHE137 CG −7.7939.34 −22.287 1004 PHEI37 CD1 −7.236 10.527 −22.776 1005 PHE137 CD2−7.258 8.117 −22.706 1006 PHE137 CE1 −6.147 10.489 −23.643 1007 PHE137CE2 −6.162 8.083 −23.57 1008 PHE137 CZ −5.604 9.269 −24.035 1009 TRP138N −11.51 7.859 −20.254 1010 TRP138 CA −12.345 7.665 −19.064 1011 TRP138C −11.447 7.497 −17.827 1012 TRP138 O −10.396 6.86 −17.86 1013 TRP138 CB−13.233 6.421 −19.182 1014 TRP138 CG −14.47 6.651 −19.997 1015 TRP138CD1 −14.621 6.385 −21.343 1016 TRP138 CD2 −15.731 7.146 −19.529 1017TRP138 NE1 −15.902 6.7 −21.705 1018 TRP138 CE2 −16.607 7.155 −20.6161019 TRP138 CE3 −16.213 7.579 −18.276 1020 TRP138 CZ2 −17.942 7.566−20.511 1021 TRP138 CZ3 −17.545 7.994 −18.155 1022 TRP138 CH2 −18.3957.984 −19.257 1023 PHE139 N −11.973 8.052 −16.675 1024 PHE139 CA −11.3067.973 −15.369 1025 PHE139 C −12.384 7.696 −14.306 1026 PHE139 O −13.5388.118 −14.423 1027 PHE139 CB −10.641 9.303 −14.98 1028 PHE139 CG −9.3319.613 −15.658 1029 PHE139 CD1 −9.286 10.091 −16.972 1030 PHE139 CD2−8.136 9.507 −14.936 1031 PHE139 CE1 −8.072 10.481 −17.539 1032 PHE139CE2 −6.928 9.916 −15.495 1033 PHE139 CZ −6.896 10.405 −16.798 1034TRP140 N −11.939 7.013 −13.188 1035 TRP140 CA −12.701 7.014 −11.929 1036TRP140 C −12.057 8.105 −11.072 1037 TRP140 O −10.834 8.217 −11.006 1038TRP140 CB −12.571 5.667 −11.208 1039 TRP140 CG −13.383 5.54 −9.948 1040TRP140 CD1 −14.64 4.977 −9.842 1041 TRP140 CD2 −12.984 5.899 −8.615 1042TRP140 NE1 −14.998 4.949 −8.521 1043 TRP140 CE2 −14.019 5.533 −7.7531044 TRP140 CE3 −11.825 6.474 −8.053 1045 TRP140 CZ2 −13.959 5.73 −6.3671046 TRP140 CZ3 −11.752 6.684 −6.671 1047 TRP140 CH2 −12.806 6.317−5.841 1048 HIS141 N −12.917 8.884 −10.331 1049 HIS141 CA −12.408 9.837−9.352 1050 HIS141 C −13.458 10.025 −8.248 1051 HIS141 O −14.635 10.302−8.48 1052 HIS141 CB −12.141 11.23 −9.934 1053 HIS141 CG −11.032 11.334−10.919 1054 HIS141 ND1 −9.75 10.877 −10.725 1055 HIS141 CD2 −11.00511.901 −12.174 1056 HIS141 CE1 −9.024 11.237 −11.83 1057 HIS141 NE2−9.754 11.864 −12.721 1058 TYR142 N −12.985 9.871 −6.958 1059 TYR142 CA−13.632 10.526 −5.814 1060 TYR142 C −15.113 10.164 −5.57 1061 TYR142 O−15.861 10.868 −4.883 1062 TYR142 CB −13.428 12.057 −5.844 1063 TYR142CG −11.989 12.464 −5.617 1064 TYR142 CD1 −11.292 13.263 −6.54 1065TYR142 CD2 −11.335 12.063 −4.448 1066 TYR142 CE1 −9.96 13.636 −6.3061067 TYR142 CE2 −10.014 12.428 −4.224 1068 TYR142 CZ −9.338 13.215−5.142 1069 TYR142 OH −8.073 13.56 −4.798 1070 GLY143 N −15.476 8.919−6.043 1071 GLY143 CA −16.832 8.412 −5.971 1072 GLY143 C −17.655 8.645−7.241 1073 GLY143 O −18.883 8.482 −7.266 1074 ASN144 N −16.955 8.951−8.384 1075 ASN144 CA −17.596 9.151 −9.681 1076 ASN144 C −16.688 8.618−10.801 1077 ASN144 O −15.464 8.573 −10.704 1078 ASN144 CB −17.922 10.63−9.913 1079 ASN144 CG −19.33 11.081 −9.576 1080 ASN144 OD1 −19.77412.166 −9.944 1081 ASN144 ND2 −20.118 10.253 −8.821 1082 TRP145 N−17.387 8.237 −11.939 1083 TRP145 CA −16.681 8.01 −13.204 1084 TRP145 C−16.708 9.355 −13.94 1085 TRP145 O −17.634 10.157 −13.793 1086 TRP145 CB−17.356 6.938 −14.068 1087 TRP145 CG −16.948 5.548 −13.681 1088 TRP145CD1 −17.601 4.704 −12.805 1089 TRP145 CD2 −15.793 4.843 −14.152 1090TRP145 NE1 −16.89 3.535 −12.723 1091 TRP145 CE2 −15.768 3.603 −13.5151092 TRP145 CE3 −14.756 5.154 −15.056 1093 TRP145 CZ2 −14.741 2.671−13.713 1094 TRP145 CZ3 −13.723 4.237 −15.269 1095 TRP145 CH2 −13.7153.018 −14.599 1096 VAL146 N −15.638 9.562 −14.788 1097 VAL146 CA −15.3910.871 −15.385 1098 VAL146 C −14.852 10.644 −16.812 1099 VAL146 O−13.837 9.963 −17.003 1100 VAL146 CB −14.358 11.689 −14.569 1101 VAL146CG1 −14.207 13.097 −15.155 1102 VAL146 CG2 −14.719 11.766 −13.081 1103PRO147 N −15.549 11.254 −17.843 1104 PRO147 CA −15.043 11.286 −19.2151105 PRO147 C −14.214 12.571 −19.39 1106 PRO147 O −14.723 13.693 −19.3731107 PRO147 CB −16.31 11.349 −20.067 1108 PRO147 CG −17.313 12.106 −19.21109 PRO147 CD −16.917 11.758 −17.771 1110 VAL148 N −12.848 12.386−19.479 1111 VAL148 CA −11.973 13.532 −19.714 1112 VAL148 C −11.75813.62 −21.235 1113 VAL148 O −11.456 12.641 −21.92 1114 VAL148 CB −10.6313.404 −18.97 1115 VAL148 CG1 −9.735 14.62 −19.234 1116 VAL148 CG2−10.856 13.273 −17.459 1117 VAL149 N −11.918 14.896 −21.747 1118 VAL149CA −11.758 15.208 −23.164 1119 VAL149 C −10.691 16.306 −23.249 1120VAL149 O −10.646 17.256 −22.459 1121 VAL149 CB −13.078 15.659 −23.8091122 VAL149 CG1 −12.86 15.9 −25.303 1123 VAL149 CG2 −14.181 14.618−23.583 1124 ILE150 N −9.774 16.139 −24.272 1125 ILE150 CA −8.459 16.763−24.18 1126 ILE150 C −7.864 16.991 −25.584 1127 ILE150 O −8.123 16.277−26.552 1128 ILE150 CB −7.579 15.931 −23.204 1129 ILE150 CG1 −7.16414.539 −23.729 1130 ILE150 CG2 −6.394 16.721 −22.679 1131 ILE150 CD1−5.892 14.481 −24.561 1132 ASP151 N −7.006 18.07 −25.645 1133 ASP151 CA−6.201 18.426 −26.811 1134 ASP151 C −4.805 17.775 −26.706 1135 ASP151 O−4.388 17.224 −25.683 1136 ASP151 CB −6.032 19.945 −26.887 1137 ASP151CG −5.19 20.42 −25.713 1138 ASP151 OD1 −4.284 21.264 −25.964 1139 ASP151OD2 −5.384 19.863 −24.585 1140 ASP152 N −4.025 17.991 −27.816 1141ASP152 CA −2.677 17.478 −27.987 1142 ASP152 C −1.662 18.603 −28.256 1143ASP152 O −0.641 18.39 −28.91 1144 ASP152 CB −2.65 16.414 −29.084 1145ASP152 CG −3.094 16.94 −30.439 1146 ASP152 OD1 −2.806 16.218 −31.4411147 ASP152 OD2 −3.733 18.037 −30.439 1148 ARG153 N −1.855 19.793−27.564 1149 ARG153 CA −0.747 20.739 −27.456 1150 ARG153 C 0.246 20.139−26.438 1151 ARG153 O −0.025 20.021 −25.241 1152 ARG153 CB −1.151 22.137−26.954 1153 ARG153 CG −1.674 23.084 −28.031 1154 ARG153 CD −3.15622.953 −28.326 1155 ARG153 NE −3.946 24.076 −27.803 1156 ARG153 CZ−4.465 24.31 −26.588 1157 ARG153 NH1 −4.293 23.508 −25.537 1158 ARG153NH2 −5.257 25.397 −26.457 1159 LEU154 N 1.429 19.688 −26.982 1160 LEU154CA 2.505 19.144 −26.165 1161 LEU154 C 3.541 20.264 −25.943 1162 LEU154 O3.869 21.026 −26.862 1163 LEU154 CB 3.194 17.955 −26.85 1164 LEU154 CG2.267 16.758 −27.143 1165 LEU154 CD1 3.043 15.674 −27.891 1166 LEU154CD2 1.648 16.167 −25.875 1167 PRO155 N 4.111 20.347 −24.685 1168 PRO155CA 5.113 21.362 −24.364 1169 PRO155 C 6.512 20.815 −24.687 1170 PRO155 O6.887 19.688 −24.347 1171 PRO155 CB 4.968 21.566 −22.858 1172 PRO155 CG4.548 20.186 −22.366 1173 PRO155 CD 3.654 19.668 −23.481 1174 VAL156 N7.327 21.709 −25.354 1175 VAL156 CA 8.68 21.349 −25.743 1176 VAL156 C9.633 22.52 −25.452 1177 VAL156 O 9.287 23.695 −25.577 1178 VAL156 CB8.788 20.883 −27.216 1179 VAL156 CG1 7.781 19.785 −27.562 1180 VAL156CG2 8.635 22.013 −28.231 1181 ASN157 N 10.914 22.138 −25.11 1182 ASN157CA 12.032 23.084 −25.07 1183 ASN157 C 12.942 22.663 −26.224 1184 ASN157O 13.399 21.524 −26.322 1185 ASN157 CB 12.82 23.005 −23.768 1186 ASN157CG 12.778 24.303 −22.999 1187 ASN157 OD1 12.36 24.37 −21.846 1188 ASN157ND2 13.288 25.39 −23.643 1189 GLU158 N 13.114 23.635 −27.193 1190 GLU158CA 14.111 23.471 −28.257 1191 GLU158 C 13.939 22.143 −29.043 1192 GLU158O 14.864 21.58 −29.619 1193 GLU158 CB 15.539 23.711 −27.735 1194 GLU158CG 15.875 25.194 −27.503 1195 GLU158 CD 15.163 25.954 −26.384 1196GLU158 OE1 14.01 25.532 −26.053 1197 GLU158 OE2 15.803 26.929 −25.8811198 ALA159 N 12.617 21.738 −29.135 1199 ALA159 CA 12.147 20.492 −29.7321200 ALA159 C 12.26 19.213 −28.87 1201 ALA159 O 11.813 18.139 −29.2781202 ALA159 CB 12.681 20.149 −31.14 1203 GLY160 N 12.833 19.347 −27.6271204 GLY160 CA 12.798 18.281 −26.635 1205 GLY160 C 11.549 18.465 −25.7741206 GLY160 O 11.176 19.573 −25.389 1207 GLN161 N 10.863 17.298 −25.4971208 GLN161 CA 9.536 17.315 −24.877 1209 GLN161 C 9.627 17.448 −23.3461210 GLN161 O 10.562 16.979 −22.701 1211 GLN161 CB 8.748 16.054 −25.2711212 GLN161 CG 8.339 16.079 −26.748 1213 GLN161 CD 7.893 14.73 −27.2641214 GLN161 OE1 6.724 14.462 −27.521 1215 GLN161 NE2 8.897 13.818−27.434 1216 LEU162 N 8.539 18.094 −22.773 1217 LEU162 CA 8.479 18.432−21.343 1218 LEU162 C 7.22 17.826 −20.681 1219 LEU162 O 6.667 18.336−19.709 1220 LEU162 CB 8.471 19.955 −21.132 1221 LEU162 CG 9.695 20.714−21.671 1222 LEU162 CD1 9.416 22.218 −21.608 1223 LEU162 CD2 10.96120.379 −20.884 1224 VAL163 N 6.852 16.608 −21.195 1225 VAL163 CA 5.73315.782 −20.703 1226 VAL163 C 6.309 14.359 −20.709 1227 VAL163 O 7.32314.092 −21.364 1228 VAL163 CB 4.505 16.045 −21.609 1229 VAL163 CG1 3.75114.822 −22.116 1230 VAL163 CG2 3.528 16.978 −20.898 1231 PHE164 N 5.64113.412 −19.96 1232 PHE164 CA 6.097 12.023 −19.996 1233 PHE164 C 5.69611.42 −21.353 1234 PHE164 O 4.599 11.614 −21.871 1235 PHE164 CB 5.5111.16 −18.874 1236 PHE164 CG 6.08 11.364 −17.485 1237 PHE164 CD1 5.24611.166 −16.374 1238 PHE164 CD2 7.433 11.648 −17.25 1239 PHE164 CE1 5.73911.272 −15.071 1240 PHE164 CE2 7.928 11.755 −15.947 1241 PHE164 CZ 7.08111.568 −14.858 1242 VAL165 N 6.676 10.652 −21.956 1243 VAL165 CA 6.61710.292 −23.37 1244 VAL165 C 7.345 8.961 −23.613 1245 VAL165 O 8.1268.471 −22.805 1246 VAL165 CB 7.223 11.381 −24.302 1247 VAL165 CG1 6.32812.612 −24.414 1248 VAL165 CG2 8.64 11.794 −23.892 1249 SER166 N 7.0798.398 −24.848 1250 SER166 CA 7.919 7.347 −25.421 1251 SER166 C 7.4987.205 −26.902 1252 SER166 O 6.799 8.053 −27.462 1253 SER166 CB 7.8776.045 −24.628 1254 SER166 OG 8.632 5.033 −25.298 1255 SER167 N 8.0796.144 −27.575 1256 SER167 CA 8.464 6.287 −28.98 1257 SER167 C 8.5734.942 −29.715 1258 SER167 O 9.355 4.741 −30.642 1259 SER167 CB 9.767.099 −29.116 1260 SER167 OG 10.789 6.523 −28.311 1261 THR168 N 7.5784.041 −29.385 1262 THR168 CA 7.352 2.822 −30.159 1263 THR168 C 5.8612.433 −30.103 1264 THR168 O 4.999 3.146 −29.584 1265 THR168 CB 8.3081.676 −29.773 1266 THR168 OG1 8.098 0.575 −30.674 1267 THR168 CG2 8.1391.18 −28.343 1268 TYR169 N 5.555 1.224 −30.708 1269 TYR169 CA 4.2350.981 −31.326 1270 TYR169 C 3.133 0.935 −30.247 1271 TYR169 O 1.9521.171 −30.479 1272 TYR169 CB 4.186 −0.38 −32.048 1273 TYR169 CG 4.901−0.542 −33.373 1274 TYR169 CD1 5.939 0.286 −33.819 1275 TYR169 CD2 4.514−1.623 −34.188 1276 TYR169 CE1 6.569 0.048 −35.046 1277 TYR169 CE2 5.137−1.86 −35.414 1278 TYR169 CZ 6.16 −1.023 −35.835 1279 TYR169 OH 6.741−1.283 −37.041 1280 LYS170 N 3.604 0.444 −29.05 1281 LYS170 CA 2.7980.211 −27.851 1282 LYS170 C 2.744 1.483 −26.973 1283 LYS170 O 1.97 1.612−26.026 1284 LYS170 CB 3.513 −0.903 −27.078 1285 LYS170 CG 2.592 −1.758−26.213 1286 LYS170 CD 3.445 −2.788 −25.473 1287 LYS170 CE 2.645 −3.746−24.614 1288 LYS170 NZ 3.578 −4.381 −23.651 1289 ASN171 N 3.768 2.368−27.234 1290 ASN171 CA 4.157 3.465 −26.36 1291 ASN171 C 3.766 4.86−26.88 1292 ASN171 O 3.772 5.831 −26.123 1293 ASN171 CB 5.673 3.426−26.177 1294 ASN171 CG 6.106 2.591 −24.996 1295 ASN171 OD1 6.751 1.552−25.119 1296 ASN171 ND2 5.736 3.079 −23.775 1297 LEU172 N 3.56 4.976−28.242 1298 LEU172 CA 3.524 6.295 −28.877 1299 LEU172 C 2.334 7.141−28.378 1300 LEU172 O 1.156 6.825 −28.533 1301 LEU172 CB 3.413 6.16−30.41 1302 LEU172 CG 4.79 6.179 −31.103 1303 LEU172 CD1 4.705 5.551−32.493 1304 LEU172 CD2 5.329 7.605 −31.219 1305 PHE173 N 2.709 8.282−27.695 1306 PHE173 CA 1.821 9.402 −27.38 1307 PHE173 C 0.666 9.132−26.405 1308 PHE173 O 0.273 10.019 −25.637 1309 PHE173 CB 1.276 10.121−28.626 1310 PHE173 CG 2.341 10.844 −29.423 1311 PHE173 CD1 2.506 10.572−30.787 1312 PHE173 CD2 3.148 11.827 −28.829 1313 PHE173 CE1 3.46611.258 −31.533 1314 PHE173 CE2 4.118 12.501 −29.574 1315 PHE173 CZ 4.27412.219 −30.927 1316 TRP174 N 0.023 7.916 −26.48 1317 TRP174 CA −1.297.75 −25.846 1318 TRP174 C −1.183 8.012 −24.328 1319 TRP174 O −2.0978.524 −23.68 1320 TRP174 CB −1.927 6.381 −26.136 1321 TRP174 CG −1.2115.23 −25.501 1322 TRP174 CD1 −0.168 4.507 −26.045 1323 TRP174 CD2 −1.4614.683 −24.201 1324 TRP174 NE1 0.247 3.582 −25.124 1325 TRP174 CE2 −0.4823.72 −23.964 1326 TRP174 CE3 −2.406 4.949 −23.187 1327 TRP174 CZ2 −0.3673.063 −22.735 1328 TRP174 CZ3 −2.329 4.256 −21.975 1329 TRP174 CH2 −1.313.34 −21.745 1330 GLY175 N 0.002 7.587 −23.752 1331 GLY175 CA 0.2367.682 −22.325 1332 GLY175 C 0.629 9.101 −21.926 1333 GLY175 O 0.44 9.536−20.791 1334 ALA176 N 1.254 9.821 −22.928 1335 ALA176 CA 1.572 11.239−22.756 1336 ALA176 C 0.261 12.029 −22.715 1337 ALA176 O 0.075 12.979−21.955 1338 ALA176 CB 2.429 11.734 −23.913 1339 LEU177 N −0.676 11.617−23.645 1340 LEU177 CA −1.98 12.249 −23.72 1341 LEU177 C −2.785 11.896−22.451 1342 LEU177 O −3.58 12.7 −21.959 1343 LEU177 CB −2.706 11.834−25 1344 LEU177 CG −2.049 12.401 −26.277 1345 LEU177 CD1 −2.554 11.649−27.507 1346 LEU177 CD2 −2.312 13.898 −26.435 1347 LEU178 N −2.59210.625 −21.934 1348 LEU178 CA −3.28 10.202 −20.707 1349 LEU178 C −2.70710.975 −19.493 1350 LEU178 O −3.431 11.346 −18.565 1351 LEU178 CB −3.1538.688 −20.509 1352 LEU178 CG −3.953 8.098 −19.331 1353 LEU178 CD1 −5.4498.386 −19.425 1354 LEU178 CD2 −3.751 6.582 −19.292 1355 GLU179 N −1.33711.179 −19.493 1356 GLU179 CA −0.666 11.977 −18.452 1357 GLU179 C −1.26913.394 −18.502 1358 GLU179 O −1.645 13.991 −17.49 1359 GLU179 CB 0.85511.976 −18.681 1360 GLU179 CG 1.656 12.821 −17.692 1361 GLU179 CD 2.2814.011 −18.424 1362 GLU179 OE1 3.48 13.853 −18.808 1363 GLU179 OE2 1.51415.003 −18.568 1364 LYS180 N −1.378 13.922 −19.776 1365 LYS180 CA −1.85615.282 −19.97 1366 LYS180 C −3.353 15.369 −19.581 1367 LYS180 O −3.84716.402 −19.118 1368 LYS180 CB −1.6 15.696 −21.418 1369 LYS180 CG −2.03917.119 −21.728 1370 LYS180 CD −1.673 17.5 −23.169 1371 LYS180 CE −2.37118.765 −23.644 1372 LYS180 NZ −3.814 18.529 −23.623 1373 ALA181 N −4.11714.246 −19.843 1374 ALA181 CA −5.532 14.194 −19.486 1375 ALA181 C −5.6914.205 −17.958 1376 ALA181 O −6.638 14.765 −17.406 1377 ALA181 CB −6.19912.946 −20.04 1378 TYR182 N −4.741 13.478 −17.262 1379 TYR182 CA −4.74713.443 −15.796 1380 TYR182 C −4.457 14.878 −15.312 1381 TYR182 O −5.09515.396 −14.394 1382 TYR182 CB −3.706 12.448 −15.261 1383 TYR182 CG−3.807 11.985 −13.822 1384 TYR182 CD1 −2.727 11.247 −13.3 1385 TYR182CD2 −4.931 12.18 −13.008 1386 TYR182 CE1 −2.776 10.707 −12.013 1387TYR182 CE2 −4.982 11.63 −11.721 1388 TYR182 CZ −3.915 10.88 −11.242 1389TYR182 OH −3.965 10.288 −10.012 1390 ALA183 N −3.434 15.531 −15.988 1391ALA183 CA −3.038 16.884 −15.608 1392 ALA183 C −4.202 17.867 −15.839 1393ALA183 O −4.4 18.834 −15.1 1394 ALA183 CB −1.832 17.348 −16.408 1395LYS184 N 4.981 17.64 −16.97 1396 LYS184 CA −5.99 18.628 −17.348 1397LYS184 C −7.037 18.763 −16.233 1398 LYS184 O −7.581 19.841 −15.989 1399LYS184 CB −6.679 18.274 −18.681 1400 LYS184 CG −7.882 19.184 −18.9791401 LYS184 CD −8.472 18.994 −20.377 1402 LYS184 CE −9.794 19.745−20.487 1403 LYS184 NZ −10.324 19.656 −21.85 1404 LEU185 N −7.438 17.574−15.652 1405 LEU185 CA −8.502 17.579 −14.661 1406 LEU185 C −7.993 18.015−13.276 1407 LEU185 O −8.758 18.521 −12.443 1408 LEU185 CB −9.19 16.216−14.574 1409 LEU185 CG −10.637 16.334 −14.048 1410 LEU185 CD1 −11.59616.865 −15.116 1411 LEU185 CD2 −11.116 14.98 −13.546 1412 SER186 N−6.679 17.712 −12.974 1413 SER186 CA −6.082 18.2 −11.73 1414 SER186 C−5.982 19.731 −11.809 1415 SER186 O −6.408 20.446 −10.901 1416 SER186 CB−4.744 17.544 −11.396 1417 SER186 OG −3.726 17.894 −12.319 1418 GLY187 N−5.389 20.215 −12.954 1419 GLY187 CA −5.295 21.628 −13.244 1420 GLY187 C−4.073 21.958 −14.088 1421 GLY187 O −4.101 22.845 −14.942 1422 SER188 N−2.929 21.274 −13.737 1423 SER188 CA −1.635 21.633 −14.299 1424 SER188 C−0.634 20.464 −14.245 1425 SER188 O −0.86 19.395 −13.679 1426 SER188 CB−1.08 22.931 −13.683 1427 SER188 OG −1.317 23.08 −12.285 1428 TYR189 N0.559 20.686 −14.923 1429 TYR189 CA 1.584 19.634 −14.924 1430 TYR189 C2.307 19.583 −13.564 1431 TYR189 O 2.837 18.545 −13.161 1432 TYR189 CB2.64 19.809 −16.024 1433 TYR189 CG 2.104 19.641 −17.427 1434 TYR189 CD12.367 20.614 −18.397 1435 TYR189 CD2 1.388 18.501 −17.808 1436 TYR189CE1 1.864 20.5 −19.69 1437 TYR189 CE2 0.894 18.376 −19.107 1438 TYR189CZ 1.125 19.379 −20.036 1439 TYR189 OH 0.641 19.22 −21.301 1440 GLU190 N2.384 20.786 −12.876 1441 GLU190 CA 3.168 20.899 −11.632 1442 GLU190 C2.609 19.903 −10.583 1443 GLU190 O 3.317 19.387 −9.715 1444 GLU190 CB3.086 22.345 −11.112 1445 GLU190 CG 3.854 22.683 −9.83 1446 GLU190 CD5.371 22.781 −9.988 1447 GLU190 OE1 5.914 21.822 −10.607 1448 GLU190 OE25.92 23.794 −9.457 1449 ASP191 N 1.247 19.682 −10.688 1450 ASP191 CA0.426 19.049 −9.662 1451 ASP191 C 0.887 17.614 −9.366 1452 ASP191 O0.648 17.058 −8.296 1453 ASP191 CB −1.033 18.939 −10.123 1454 ASP191 CG−1.597 20.089 −10.935 1455 ASP191 OD1 −2.749 19.891 −11.438 1456 ASP191OD2 −0.842 21.096 −11.106 1457 LEU192 N 1.439 16.958 −10.45 1458 LEU192CA 1.598 15.508 −10.482 1459 LEU192 C 2.801 15.062 −9.63 1460 LEU192 O2.951 13.901 −9.246 1461 LEU192 CB 1.795 15.005 −11.92 1462 LEU192 CG0.622 15.292 −12.882 1463 LEU192 CD1 0.972 14.781 −14.281 1464 LEU192CD2 −0.69 14.659 −12.415 1465 GLN193 N 3.748 16.036 −9.4 1466 GLN193 CA4.968 15.752 −8.659 1467 GLN193 C 4.574 15.629 −7.176 1468 GLN193 O4.262 16.607 −6.498 1469 GLN193 CB 5.987 16.876 −8.833 1470 GLN193 CG6.408 17.051 −10.293 1471 GLN193 CD 6.947 18.435 −10.567 1472 GLN193 OE18.067 18.62 −11.029 1473 GLN193 NE2 6.061 19.431 −10.274 1474 SER194 N4.539 14.329 −6.718 1475 SER194 CA 3.995 13.858 −5.439 1476 SER194 C2.514 13.433 −5.505 1477 SER194 O 1.863 13.182 −4.484 1478 SER194 CB4.308 14.701 −4.189 1479 SER194 OG 3.332 15.722 −3.907 1480 GLY195 N2.041 13.202 −6.782 1481 GLY195 CA 1.101 12.127 −7.04 1482 GLY195 C1.928 10.845 −7.219 1483 GLY195 O 3.15 10.857 −7.377 1484 GLN196 N 1.1869.688 −7.163 1485 GLN196 CA 1.797 8.377 −7.026 1486 GLN196 C 0.965 7.331−7.793 1487 GLN196 O −0.253 7.41 −7.969 1488 GLN196 CB 1.808 7.923−5.553 1489 GLN196 CG 2.661 8.755 −4.601 1490 GLN196 CD 2.095 8.793−3.196 1491 GLN196 OE1 1.964 9.849 −2.572 1492 GLN196 NE2 1.777 7.59−2.63 1493 VAL197 N 1.681 6.168 −8.084 1494 VAL197 CA 1.01 5.06 −8.7741495 VAL197 C −0.116 4.492 −7.891 1496 VAL197 O −1.095 3.91 −8.354 1497VAL197 CB 1.965 3.914 −9.166 1498 VAL197 CG1 2.893 4.344 −10.297 1499VAL197 CG2 2.792 3.356 −8.003 1500 SER198 N 0.102 4.647 −6.537 1501SER198 CA −0.789 4.109 −5.523 1502 SER198 C −2.164 4.782 −5.597 1503SER198 O −3.17 4.207 −5.178 1504 SER198 CB −0.199 4.308 −4.12 1505SER198 OG 0.213 5.664 −3.897 1506 GLU199 N −2.127 6.1 −6.024 1507 GLU199CA −3.344 6.848 −6.325 1508 GLU199 C −3.753 6.559 −7.786 1509 GLU199 O−4.916 6.304 −8.108 1510 GLU199 CB −3.127 8.357 −6.167 1511 GLU199 CG−2.568 8.791 −4.805 1512 GLU199 CD −1.449 9.818 −4.893 1513 GLU199 OE1−0.876 9.96 −6.007 1514 GLU199 OE2 −1.141 10.415 −3.814 1515 ALA200 N−2.74 6.657 −8.734 1516 ALA200 CA −3.103 6.771 −10.149 1517 ALA200 C−3.797 5.49 −10.633 1518 ALA200 O −4.67 5.494 −11.502 1519 ALA200 CB−1.874 7.023 −11.007 1520 LEU201 N −3.322 4.319 −10.067 1521 LEU201 CA−3.888 3.038 −10.447 1522 LEU201 C −5.325 2.882 −9.906 1523 LEU201 O−6.103 2.079 −10.432 1524 LEU201 CB −3.011 1.864 −9.998 1525 LEU201 CG−1.642 1.78 −10.709 1526 LEU201 CD1 −0.767 0.732 −10.018 1527 LEU201 CD2−1.779 1.433 −12.193 1528 VAL202 N −5.667 3.646 −8.801 1529 VAL202 CA−7.065 3.737 −8.368 1530 VAL202 C −7.818 4.513 −9.456 1531 VAL202 O−8.867 4.067 −9.927 1532 VAL202 CB −7.246 4.416 −6.992 1533 VAL202 CG1−8.72 4.645 −6.65 1534 VAL202 CG2 −6.578 3.6 −5.892 1535 ASP203 N −7.2545.713 −9.85 1536 ASP203 CA −8.006 6.652 −10.692 1537 ASP203 C −8.3975.992 −12.047 1538 ASP203 O −9.411 6.314 −12.673 1539 ASP203 CB −7.1877.906 −10.97 1540 ASP203 CG −7.174 8.932 −9.842 1541 ASP203 OD1 −8.2939.238 −9.338 1542 ASP203 OD2 −6.035 9.446 −9.607 1543 PHE204 N −7.4785.086 −12.557 1544 PHE204 CA −7.72 4.42 −13.845 1545 PHE204 C −8.6533.196 −13.671 1546 PHE204 O −9.343 2.734 −14.585 1547 PHE204 CB −6.4183.901 −14.479 1548 PHE204 CG −5.392 4.96 −14.801 1549 PHE204 CD1 −5.7216.123 −15.505 1550 PHE204 CD2 −4.062 4.771 −14.4 1551 PHE204 CE1 −4.7477.093 −15.756 1552 PHE204 CE2 −3.099 5.749 −14.641 1553 PHE204 CZ −3.4396.913 −15.318 1554 THR205 N −8.498 2.525 −12.471 1555 THR205 CA −9.1811.26 −12.237 1556 THR205 C −10.57 1.499 −11.629 1557 THR205 O −11.5590.925 −12.094 1558 THR205 CB −8.375 0.292 −11.355 1559 THR205 OG1 −7.0230.205 −11.813 1560 THR205 CG2 −8.953 −1.117 −11.399 1561 GLY206 N −10.572.248 −10.471 1562 GLY206 CA −11.736 2.417 −9.612 1563 GLY206 C −11.7731.408 −8.46 1564 GLY206 O −12.704 1.354 −7.66 1565 GLY207 N −10.6740.575 −8.411 1566 GLY207 CA −10.481 −0.4 −7.359 1567 GLY207 C −9.7010.191 −6.19 1568 GLY207 O −9.342 1.364 −6.144 1569 VAL208 N −9.446−0.722 −5.185 1570 VAL208 CA −8.721 −0.33 −3.977 1571 VAL208 C −7.241−0.71 −4.177 1572 VAL208 O −6.892 −1.808 −4.614 1573 VAL208 CB −9.326−1.027 −2.736 1574 VAL208 CG1 −8.569 −0.683 −1.451 1575 VAL208 CG2−10.802 −0.645 −2.557 1576 THR209 N −6.341 0.271 −3.793 1577 THR209 CA−4.903 −0.012 −3.717 1578 THR209 C −4.681 −0.655 −2.348 1579 THR209 O−5.049 −0.111 −1.308 1580 THR209 CB −4.045 1.266 −3.824 1581 THR209 OG1−4.047 1.727 −5.178 1582 THR209 CG2 −2.587 1.029 −3.45 1583 MET210 N−4.037 −1.872 −2.386 1584 MET210 CA −3.614 −2.558 −1.167 1585 MET210 C−2.079 −2.547 −1.22 1586 MET210 O −1.446 −3.248 −2.012 1587 MET210 CB−4.185 −3.983 −1.139 1588 MET210 CG −4.994 −4.283 0.122 1589 MET210 SD−3.932 −4.483 1.581 1590 MET210 CE −5.23 −4.677 2.828 1591 THR211 N−1.497 −1.595 −0.405 1592 THR211 CA −0.058 −1.513 −0.171 1593 THR211 C0.329 −2.517 0.925 1594 THR211 O −0.404 −2.788 1.874 1595 THR211 CB0.362 −0.109 0.315 1596 THR211 OG1 −0.653 0.417 1.181 1597 THR211 CG20.568 0.868 −0.834 1598 ILE212 N 1.606 −3.032 0.784 1599 ILE212 CA 2.161−3.992 1.731 1600 ILE212 C 3.653 −3.623 1.842 1601 ILE212 O 4.439 −3.6790.89 1602 ILE212 CB 1.996 −5.467 1.269 1603 ILE212 CG1 0.544 −5.7870.842 1604 ILE212 CG2 2.459 −6.409 2.391 1605 ILE212 CD1 0.323 −7.2240.389 1606 ASN213 N 4.051 −3.196 3.097 1607 ASN213 CA 5.445 −2.852 3.3771608 ASN213 C 6.198 −4.176 3.565 1609 ASN213 O 6.374 −4.712 4.655 1610ASN213 CB 5.591 −1.95 4.597 1611 ASN213 CG 5.543 −0.495 4.194 1612ASN213 OD1 4.56 0.217 4.374 1613 ASN213 ND2 6.679 −0.011 3.606 1614LEU214 N 6.631 −4.743 2.372 1615 LEU214 CA 6.849 −6.196 2.257 1616LEU214 C 7.948 −6.786 3.164 1617 LEU214 O 8.071 −8 3.325 1618 LEU214 CB7.21 −6.524 0.797 1619 LEU214 CG 6.781 −7.924 0.322 1620 LEU214 CD15.264 −8.091 0.35 1621 LEU214 CD2 7.299 −8.169 −1.097 1622 ALA215 N8.799 −5.866 3.743 1623 ALA215 CA 9.746 −6.28 4.771 1624 ALA215 C 9.004−6.749 6.035 1625 ALA215 O 9.443 −7.658 6.743 1626 ALA215 CB 10.69−5.138 5.114 1627 GLU216 N 7.856 −6.051 6.362 1628 GLU216 CA 6.965−6.512 7.432 1629 GLU216 C 6.126 −7.656 6.824 1630 GLU216 O 4.964 −7.5356.437 1631 GLU216 CB 6.095 −5.354 7.928 1632 GLU216 CG 5.351 −5.7039.216 1633 GLU216 CD 4.479 −4.568 9.76 1634 GLU216 OE1 4.149 −4.70510.973 1635 GLU216 OE2 4.185 −3.649 8.948 1636 ALA217 N 6.837 −8.8456.702 1637 ALA217 CA 6.304 −9.929 5.891 1638 ALA217 C 5.088 −10.5226.619 1639 ALA217 O 5.13 −10.958 7.768 1640 ALA217 CB 7.341 −11.0175.666 1641 HIS218 N 3.921 −10.47 5.876 1642 HIS218 CA 2.657 −10.92 6.4461643 HIS218 C 2.558 −12.444 6.27 1644 HIS218 O 3.016 −13.043 5.297 1645HIS218 CB 1.464 −10.242 5.762 1646 HIS218 CG 1.262 −8.812 6.152 1647HIS218 ND1 2.268 −7.905 6.403 1648 HIS218 CD2 0.101 −8.086 6.327 1649HIS218 CE1 1.678 −6.716 6.736 1650 HIS218 NE2 0.368 −6.792 6.683 1651GLY219 N 1.808 −13.081 7.253 1652 GLY219 CA 1.951 −14.522 7.447 1653GLY219 C 1.238 −15.416 6.431 1654 GLY219 O 1.25 −16.64 6.533 1655 ASN220N 0.606 −14.742 5.413 1656 ASN220 CA −0.097 −15.41 4.321 1657 ASN220 C0.08 −14.582 3.033 1658 ASN220 O −0.798 −14.425 2.188 1659 ASN220 CB−1.55 −15.706 4.677 1660 ASN220 CG −2.411 −14.472 4.791 1661 ASN220 OD1−3.291 −14.195 3.978 1662 ASN220 ND2 −2.161 −13.678 5.875 1663 LEU221 N1.381 −14.127 2.838 1664 LEU221 CA 1.657 −13.17 1.758 1665 LEU221 C1.537 −13.871 0.395 1666 LEU221 O 1.097 −13.298 −0.603 1667 LEU221 CB3.045 −12.546 1.961 1668 LEU221 CG 3.463 −11.398 1.019 1669 LEU221 CD14.053 −11.903 −0.296 1670 LEU221 CD2 2.349 −10.38 0.782 1671 TRP222 N2.049 −15.157 0.345 1672 TRP222 CA 1.983 −15.898 −0.921 1673 TRP222 C0.519 −16.115 −1.317 1674 TRP222 O 0.144 −16.108 −2.489 1675 TRP222 CB2.717 −17.243 −0.832 1676 TRP222 CG 3.702 −17.433 −1.948 1677 TRP222 CD13.681 −18.413 −2.923 1678 TRP222 CD2 4.845 −16.612 −2.211 1679 TRP222NE1 4.778 −18.245 −3.728 1680 TRP222 CE2 5.502 −17.153 −3.312 1681TRP222 CE3 5.376 −15.448 −1.617 1682 TRP222 CZ2 6.677 −16.608 −3.8311683 TRP222 CZ3 6.521 −14.861 −2.163 1684 TRP222 CH2 7.167 −15.439−3.249 1685 ASP223 N −0.329 −16.336 −0.26 1686 ASP223 CA −1.732 −16.674−0.436 1687 ASP223 C −2.439 −15.457 −1.071 1688 ASP223 O −3.327 −15.585−1.915 1689 ASP223 CB −2.365 −17.05 0.899 1690 ASP223 CG −1.492 −17.9361.793 1691 ASP223 OD1 −2.122 −18.723 2.549 1692 ASP223 OD2 −0.241−17.716 1.707 1693 ILE224 N −2.011 −14.218 −0.61 1694 ILE224 CA −2.557−12.964 −1.141 1695 ILE224 C −2.205 −12.885 −2.64 1696 ILE224 O −3.014−12.473 −3.475 1697 ILE224 CB −2.02 −11.714 −0.388 1698 ILE224 CG1−2.573 −11.676 1.05 1699 ILE224 CG2 −2.385 −10.409 −1.116 1700 ILE224CD1 −1.906 −10.642 1.947 1701 LEU225 N −0.9 −13.202 −2.961 1702 LEU225CA −0.423 −13.139 −4.339 1703 LEU225 C −1.047 −14.261 −5.206 1704 LEU225O −1.184 −14.141 −6.428 1705 LEU225 CB 1.105 −13.173 −4.41 1706 LEU225CG 1.79 −11.79 −4.36 1707 LEU225 CD1 1.338 −10.9 −3.204 1708 LEU225 CD23.308 −11.981 −4.304 1709 ILE226 N −1.398 −15.419 −4.543 1710 ILE226 CA−2.158 −16.482 −5.211 1711 ILE226 C −3.569 −15.908 −5.494 1712 ILE226 O−4.099 −16.024 −6.603 1713 ILE226 CB −2.153 −17.799 −4.401 1714 ILE226CG1 −0.769 −18.483 −4.52 1715 ILE226 CG2 −3.251 −18.76 −4.869 1716ILE226 CD1 −0.545 −19.602 −3.512 1717 GLU227 N −4.195 −15.25 −4.447 1718GLU227 CA −5.531 −14.679 −4.634 1719 GLU227 C −5.479 −13.627 −5.764 1720GLU227 O −6.373 −13.499 −6.603 1721 GLU227 CB −6.066 −13.998 −3.364 1722GLU227 CG −7.271 −14.723 −2.779 1723 GLU227 CD −6.902 −15.919 −1.9091724 GLU227 OE1 −6.286 −15.606 −0.837 1725 GLU227 OE2 −7.305 −17.042−2.336 1726 ALA228 N −4.374 −12.795 −5.728 1727 ALA228 CA −4.271 −11.646−6.618 1728 ALA228 C −4.181 −12.118 −8.08 1729 ALA228 O −4.639 −11.458−9.016 1730 ALA228 CB −3.029 −10.841 −6.274 1731 THR229 N −3.461 −13.284−8.258 1732 THR229 CA −3.262 −13.877 −9.57 1733 THR229 C −4.478 −14.672−10.083 1734 THR229 O −4.599 −14.866 −11.298 1735 THR229 CB −1.972−14.717 −9.672 1736 THR229 OG1 −1.776 −15.533 −8.52 1737 THR229 CG2−0.729 −13.851 −9.858 1738 TYR230 N −5.375 −15.178 −9.154 1739 TYR230 CA−6.665 −15.726 −9.594 1740 TYR230 C −7.839 −14.716 −9.578 1741 TYR230 O−8.954 −15.009 −10.022 1742 TYR230 CB −7.009 −17.12 −9.043 1743 TYR230CG −7.495 −17.25 −7.62 1744 TYR230 CD1 −6.708 −17.904 −6.661 1745 TYR230CD2 −8.779 −16.824 −7.252 1746 TYR230 CE1 −7.187 −18.111 −5.364 1747TYR230 CE2 −9.252 −17.022 −5.955 1748 TYR230 CZ −8.453 −17.658 −5.021749 TYR230 OH −8.933 −17.814 −3.747 1750 ASN231 N −7.53 −13.45 −9.121751 ASN231 CA −8.366 −12.287 −9.435 1752 ASN231 C −7.876 −11.655−10.756 1753 ASN231 O −8.659 −11.094 −11.523 1754 ASN231 CB −8.348−11.242 −8.326 1755 ASN231 CG −9.222 −10.039 −8.619 1756 ASN231 OD1−8.789 −8.888 −8.614 1757 ASN231 ND2 −10.546 −10.28 −8.837 1758 ARG232 N−6.507 −11.647 −10.936 1759 ARG232 CA −5.833 −11 −12.059 1760 ARG232 C−5.824 −9.478 −11.843 1761 ARG232 O −6.255 −8.671 −12.672 1762 ARG232 CB−6.345 −11.407 −13.448 1763 ARG232 CG −6.11 −12.893 −13.724 1764 ARG232CD −6.664 −13.312 −15.077 1765 ARG232 NE −6.351 −14.717 −15.369 1766ARG232 CZ −5.132 −15.175 −15.748 1767 ARG232 NH1 −4.95 −16.509 −15.8841768 ARG232 NH2 −4.091 −14.362 −16.01 1769 THR233 N −5.206 −9.094−10.668 1770 THR233 CA −4.818 −7.708 −10.41 1771 THR233 C −3.502 −7.41−11.169 1772 THR233 O −2.874 −8.268 −11.79 1773 THR233 CB −4.613 −7.455−8.905 1774 THR233 OG1 −3.54 −8.265 −8.436 1775 THR233 CG2 −5.843 −7.771−8.071 1776 LEU234 N −3.086 −6.084 −11.103 1777 LEU234 CA −1.673 −5.76−11.307 1778 LEU234 C −1.027 −5.808 −9.904 1779 LEU234 O −1.659 −5.515−8.885 1780 LEU234 CB −1.478 −4.37 −11.924 1781 LEU234 CG −2.119 −4.193−13.316 1782 LEU234 CD1 −1.98 −2.738 −13.772 1783 LEU234 CD2 −1.502−5.121 −14.364 1784 ILE235 N 0.303 −6.192 −9.912 1785 ILE235 CA 1.03−6.564 −8.693 1786 ILE235 C 2.451 −6.001 −8.879 1787 ILE235 O 3.222−6.474 −9.721 1788 ILE235 CB 1.117 −8.11 −8.538 1789 ILE235 CG1 −0.254−8.808 −8.634 1790 ILE235 CG2 1.846 −8.473 −7.234 1791 ILE235 CD1 −0.173−10.328 −8.602 1792 GLY236 N 2.759 −4.917 −8.08 1793 GLY236 CA 4.011−4.19 −8.235 1794 GLY236 C 4.421 −3.56 −6.91 1795 GLY236 O 3.603 −3.04−6.148 1796 CYS237 N 5.725 −3.547 −6.529 1797 CYS237 CA 6.857 −4.283−7.087 1798 CYS237 C 8.063 −3.78 −6.284 1799 CYS237 O 7.91 −3.319 −5.1531800 CYS237 CB 6.703 −5.791 −6.864 1801 CYS237 SG 7.471 −6.738 −8.1991802 GLN238 N 9.292 −3.94 −6.884 1803 GLN238 CA 10.552 −3.904 −6.1051804 GLN238 C 11.444 −4.893 −6.899 1805 GLN238 O 10.903 −5.735 −7.6261806 GLN238 CB 11.161 −2.511 −5.884 1807 GLN238 CG 10.422 −1.538 −4.9521808 GLN238 CD 10.355 −1.958 −3.489 1809 GLN238 OE1 11.034 −1.48 −2.5831810 GLN238 NE2 9.443 −2.924 −3.213 1811 THR239 N 12.807 −4.866 −6.71812 THR239 CA 13.655 −5.762 −7.512 1813 THR239 C 13.612 −7.169 −6.9011814 THR239 O 13.197 −8.155 −7.508 1815 THR239 CB 15.092 −5.205 −7.5881816 THR239 OG1 14.982 −3.829 −7.972 1817 THR239 CG2 15.953 −5.988 −8.571818 HIS240 N 14.09 −7.248 −5.61 1819 HIS240 CA 14.281 −8.527 −4.9141820 HIS240 C 12.978 −8.912 −4.203 1821 HIS240 O 12.88 −9.04 −2.985 1822HIS240 CB 15.458 −8.418 −3.936 1823 HIS240 CG 16.743 −8.341 −4.684 1824HIS240 ND1 17.417 −9.453 −5.129 1825 HIS240 CD2 17.479 −7.272 −5.1441826 HIS240 CE1 18.495 −9.005 −5.838 1827 HIS240 NE2 18.56 −7.693 −5.8651828 SER241 N 11.916 −9.112 −5.071 1829 SER241 CA 10.542 −9.313 −4.6071830 SER241 C 10.161 −10.799 −4.555 1831 SER241 O 9.027 −11.208 −4.8011832 SER241 CB 9.546 −8.498 −5.441 1833 SER241 OG 8.59 −7.843 −4.6041834 GLY242 N 11.138 −11.604 −4.005 1835 GLY242 CA 10.886 −12.991 −3.6851836 GLY242 C 11.518 −13.984 −4.646 1837 GLY242 O 11.058 −15.12 −4.7681838 GLU243 N 12.701 −13.574 −5.222 1839 GLU243 CA 13.646 −14.531 −5.7951840 GLU243 C 14.662 −14.811 −4.652 1841 GLU243 O 14.268 −14.833 −3.4741842 GLU243 CB 14.083 −14.032 −7.179 1843 GLU243 CG 12.878 −14.115−8.149 1844 GLU243 CD 13.078 −14.26 −9.661 1845 GLU243 OE1 13.914−13.477 −10.185 1846 GLU243 OE2 12.38 −15.174 −10.223 1847 LYS244 N15.967 −15.146 −4.963 1848 LYS244 CA 16.915 −15.495 −3.891 1849 LYS244 C18.043 −14.481 −3.601 1850 LYS244 O 18.714 −14.546 −2.563 1851 LYS244 CB17.58 −16.87 −4.126 1852 LYS244 CG 16.585 −18.033 −4.084 1853 LYS244 CD17.202 −19.34 −3.557 1854 LYS244 CE 16.229 −20.489 −3.773 1855 LYS244 NZ16.694 −21.744 −3.179 1856 ILE245 N 18.33 −13.6 −4.621 1857 ILE245 CA19.704 −13.1 −4.817 1858 ILE245 C 20.105 −12.091 −3.705 1859 ILE245 O19.301 −11.591 −2.912 1860 ILE245 CB 19.866 −12.645 −6.297 1861 ILE245CG1 19.798 −13.898 −7.213 1862 ILE245 CG2 21.135 −11.843 −6.591 1863ILE245 CD1 19.972 −13.612 −8.696 1864 LEU246 N 21.47 −11.909 −3.553 1865LEU246 CA 22.04 −11.035 −2.526 1866 LEU246 C 21.875 −9.555 −2.918 1867LEU246 O 21.604 −9.188 −4.057 1868 LEU246 CB 23.526 −11.357 −2.297 1869LEU246 CG 23.787 −12.752 −1.695 1870 LEU246 CD1 25.282 −13.077 −1.7621871 LEU246 CD2 23.311 −12.848 −0.244 1872 GLU247 N 22.06 −8.689 −1.8561873 GLU247 CA 21.764 −7.257 −1.941 1874 GLU247 C 22.653 −6.574 −0.8771875 GLU247 O 23.403 −7.203 −0.125 1876 GLU247 CB 20.257 −7.031 −1.6871877 GLU247 CG 19.585 −6.072 −2.672 1878 GLU247 CD 19.777 −4.615 −2.31879 GLU247 OE1 18.747 −3.984 −1.931 1880 GLU247 OE2 20.979 −4.202−2.313 1881 ASN248 N 22.551 −5.209 −0.847 1882 ASN248 CA 23.218 −4.3310.109 1883 ASN248 C 22.391 −3.063 0.424 1884 ASN248 O 22.479 −2.526 1.531885 ASN248 CB 24.616 −3.956 −0.38 1886 ASN248 CG 25.697 −4.59 0.4691887 ASN248 OD1 26.558 −3.934 1.047 1888 ASN248 ND2 25.696 −5.956 0.5151889 GLY249 N 21.675 −2.496 −0.615 1890 GLY249 CA 20.704 −1.458 −0.3261891 GLY249 C 19.996 −0.918 −1.571 1892 GLY249 O 20.588 −0.612 −2.6041893 LEU250 N 18.631 −0.761 −1.405 1894 LEU250 CA 17.733 −0.144 −2.3821895 LEU250 C 17.442 −1.024 −3.615 1896 LEU250 O 16.294 −1.159 −4.0431897 LEU250 CB 18.165 1.266 −2.829 1898 LEU250 CG 18.419 2.269 −1.6851899 LEU250 CD1 18.916 3.597 −2.264 1900 LEU250 CD2 17.174 2.512 −0.8321901 VAL251 N 18.555 −1.5 −4.273 1902 VAL251 CA 18.487 −2.051 −5.63 1903VAL251 C 18.664 −0.876 −6.614 1904 VAL251 O 19.193 0.189 −6.286 1905VAL251 CB 19.538 −3.173 −5.802 1906 VAL251 CG1 20.982 −2.666 −5.845 1907VAL251 CG2 19.264 −4.06 −7.016 1908 GLU252 N 18.244 −1.121 −7.904 1909GLU252 CA 18.313 −0.099 −8.931 1910 GLU252 C 17.064 0.805 −8.898 1911GLU252 O 16.015 0.521 −8.32 1912 GLU252 CB 18.559 −0.724 −10.313 1913GLU252 CG 17.381 −1.462 −10.947 1914 GLU252 CD 16.937 −2.788 −10.3371915 GLU252 OE1 16.583 −3.673 −11.176 1916 GLU252 OE2 16.902 −2.853−9.067 1917 GLY253 N 17.189 1.972 −9.635 1918 GLY253 CA 16.143 2.98−9.629 1919 GLY253 C 15.005 2.579 −10.561 1920 GLY253 O 14.857 3.072−11.678 1921 HIS254 N 14.186 1.593 −10.045 1922 HIS254 CA 13.164 0.948−10.863 1923 HIS254 C 12.181 0.227 −9.928 1924 HIS254 O 12.461 −0.07−8.768 1925 HIS254 CB 13.814 −0.05 −11.838 1926 HIS254 CG 13.022 −0.295−13.075 1927 HIS254 ND1 12.803 0.665 −14.031 1928 HIS254 CD2 12.416−1.427 −13.576 1929 HIS254 CE1 12.073 0.088 −15.029 1930 HIS254 NE211.843 −1.182 −14.795 1931 ALA255 N 10.976 −0.101 −10.519 1932 ALA255 CA10.04 −1.017 −9.884 1933 ALA255 C 9.562 −1.974 −10.98 1934 ALA255 O9.432 −1.627 −12.151 1935 ALA255 CB 8.88 −0.293 −9.228 1936 TYR256 N9.353 −3.256 −10.524 1937 TYR256 CA 9.061 −4.376 −11.419 1938 TYR256 C7.558 −4.671 −11.306 1939 TYR256 O 6.814 −4.032 −10.559 1940 TYR256 CB9.918 −5.601 −11.046 1941 TYR256 CG 11.385 −5.477 −11.403 1942 TYR256CD1 12.185 −4.472 −10.843 1943 TYR256 CD2 11.987 −6.408 −12.262 1944TYR256 CE1 13.533 −4.357 −11.178 1945 TYR256 CE2 13.343 −6.305 −12.5831946 TYR256 CZ 14.104 −5.269 −12.053 1947 TYR256 OH 15.418 −5.186−12.413 1948 THR257 N 7.096 −5.697 −12.106 1949 THR257 CA 5.751 −6.245−11.912 1950 THR257 C 5.882 −7.767 −11.761 1951 THR257 O 6.862 −8.39−12.172 1952 THR257 CB 4.785 −5.826 −13.033 1953 THR257 OG1 3.438 −6.226−12.762 1954 THR257 CG2 5.178 −6.346 −14.404 1955 LEU258 N 4.796 −8.365−11.156 1956 LEU258 CA 4.758 −9.801 −10.82 1957 LEU258 C 3.657 −10.421−11.703 1958 LEU258 O 2.578 −9.849 −11.876 1959 LEU258 CB 4.477 −9.917−9.316 1960 LEU258 CG 5.035 −11.17 −8.621 1961 LEU258 CD1 5.351 −10.838−7.159 1962 LEU258 CD2 4.036 −12.324 −8.672 1963 THR259 N 3.969 −11.634−12.313 1964 THR259 CA 3.099 −12.191 −13.367 1965 THR259 C 2.945 −13.737−13.36 1966 THR259 O 2.503 −14.372 −14.326 1967 THR259 CB 3.424 −11.686−14.802 1968 THR259 OG1 4.665 −12.213 −15.307 1969 THR259 CG2 3.491−10.171 −14.94 1970 GLY260 N 3.177 −14.344 −12.141 1971 GLY260 CA 3.017−15.778 −11.933 1972 GLY260 C 3.617 −16.24 −10.601 1973 GLY260 O 4.34−15.515 −9.914 1974 ILE261 N 3.277 −17.542 −10.254 1975 ILE261 CA 3.485−18.055 −8.884 1976 ILE261 C 3.328 −19.605 −8.878 1977 ILE261 O 2.85−20.201 −9.849 1978 ILE261 CB 2.487 −17.336 −7.933 1979 ILE261 CG1 2.935−17.375 −6.465 1980 ILE261 CG2 1.049 −17.849 −8.094 1981 ILE261 CD12.357 −16.213 −5.673 1982 ARG262 N 3.751 −20.227 −7.708 1983 ARG262 CA3.281 −21.535 −7.183 1984 ARG262 C 4.264 −22.043 −6.083 1985 ARG262 O5.187 −21.344 −5.659 1986 ARG262 CB 3.005 −22.6 −8.255 1987 ARG262 CG4.269 −23.015 −9.005 1988 ARG262 CD 3.951 −23.82 −10.255 1989 ARG262 NE3.381 −22.955 −11.296 1990 ARG262 CZ 3.328 −23.325 −12.598 1991 ARG262NH1 2.901 −22.461 −13.537 1992 ARG262 NH2 3.705 −24.548 −13.015 1993LYS263 N 4.005 −23.303 −5.575 1994 LYS263 CA 4.937 −24.024 −4.693 1995LYS263 C 5.465 −25.246 −5.468 1996 LYS263 O 4.778 −25.813 −6.32 1997LYS263 CB 4.195 −24.458 −3.421 1998 LYS263 CG 5.102 −25.081 −2.357 1999LYS263 CD 4.339 −25.363 −1.061 2000 LYS263 CE 5.27 −25.934 0 2001 LYS263NZ 4.644 −25.802 1.335 2002 VAL264 N 6.718 −25.681 −5.083 2003 VAL264 CA7.326 −26.916 −5.581 2004 VAL264 C 8.106 −27.592 −4.428 2005 VAL264 O8.335 −27.036 −3.352 2006 VAL264 CB 8.248 −26.692 −6.807 2007 VAL264 CG17.47 −26.229 −8.04 2008 VAL264 CG2 9.396 −25.727 −6.504 2009 THR265 N8.554 −28.869 −4.72 2010 THR265 CA 9.554 −29.535 −3.884 2011 THR265 C10.787 −29.809 −4.756 2012 THR265 O 10.694 −30.025 −5.965 2013 THR265 CB8.991 −30.793 −3.206 2014 THR265 OG1 9.877 −31.233 −2.175 2015 THR265CG2 8.73 −31.956 −4.153 2016 CYS266 N 11.978 −29.812 −4.061 2017 CYS266CA 13.284 −29.87 −4.714 2018 CYS266 C 14.097 −30.88 −3.895 2019 CYS266 O14.691 −30.581 −2.86 2020 CYS266 CB 13.989 −28.509 −4.7 2021 CYS266 SG13.206 −27.305 −5.819 2022 LYS267 N 13.961 −32.195 −4.31 2023 LYS267 CA14.689 −33.299 −3.673 2024 LYS267 C 14.286 −33.478 −2.192 2025 LYS267 O15.01 −34.029 −1.367 2026 LYS267 CB 16.213 −33.232 −3.892 2027 LYS267 CG16.663 −34.184 −5.011 2028 LYS267 CD 18.157 −34.036 −5.317 2029 LYS267CE 18.571 −34.928 −6.481 2030 LYS267 NZ 20.005 −34.709 −6.786 2031HIS268 N 12.97 −33.132 −1.95 2032 HIS268 CA 12.274 −33.231 −0.667 2033HIS268 C 12.482 −32.006 0.241 2034 HIS268 O 11.993 −31.959 1.37 2035HIS268 CB 12.498 −34.526 0.134 2036 HIS268 CG 12.281 −35.759 −0.675 2037HIS268 ND1 13.322 −36.468 −1.223 2038 HIS268 CD2 11.152 −36.446 −1.0612039 HIS268 CE1 12.789 −37.516 −1.915 2040 HIS268 NE2 11.478 −37.53−1.837 2041 ARG269 N 13.176 −30.951 −0.316 2042 ARG269 CA 13.144 −29.6180.276 2043 ARG269 C 12.021 −28.86 −0.454 2044 ARG269 O 12.043 −28.758−1.688 2045 ARG269 CB 14.46 −28.861 0.046 2046 ARG269 CG 15.662 −29.5590.684 2047 ARG269 CD 16.953 −28.794 0.405 2048 ARG269 NE 18.104 −29.4741.018 2049 ARG269 CZ 19.385 −29.046 0.903 2050 ARG269 NH1 20.373 −29.7441.502 2051 ARG269 NH2 19.706 −27.938 0.204 2052 PRO270 N 11.014 −28.2930.298 2053 PRO270 CA 9.987 −27.465 −0.337 2054 PRO270 C 10.563 −26.091−0.7 2055 PRO270 O 11.354 −25.501 0.037 2056 PRO270 CB 8.9 −27.291 0.7292057 PRO270 CG 9.144 −28.441 1.697 2058 PRO270 CD 10.654 −28.613 1.672059 GLU271 N 10.059 −25.54 −1.864 2060 GLU271 CA 10.405 −24.182 −2.2732061 GLU271 C 9.161 −23.549 −2.941 2062 GLU271 O 8.302 −24.202 −3.5342063 GLU271 CB 11.647 −24.106 −3.183 2064 GLU271 CG 12.953 −24.496−2.481 2065 GLU271 CD 14.184 −24.134 −3.291 2066 GLU271 OE1 14.842−23.104 −2.938 2067 GLU271 OE2 14.491 −24.885 −4.272 2068 TYR272 N 9.087−22.177 −2.79 2069 TYR272 CA 7.922 −21.389 −3.21 2070 TYR272 C 8.368−20.54 −4.407 2071 TYR272 O 9.178 −19.715 −4.286 2072 TYR272 CB 7.46−20.467 −2.073 2073 TYR272 CG 6.69 −21.185 −0.988 2074 TYR272 CD1 7.353−21.902 0.018 2075 TYR272 CD2 5.289 −21.136 −0.972 2076 TYR272 CE1 6.629−22.565 1.011 2077 TYR272 CE2 4.562 −21.773 0.033 2078 TYR272 CZ 5.241−22.492 1.008 2079 TYR272 OH 4.541 −23.204 1.947 2080 LEU273 N 7.728−20.835 −5.606 2081 LEU273 CA 8.076 −20.123 −6.831 2082 LEU273 C 7.317−18.786 −6.864 2083 LEU273 O 6.155 −18.677 −6.464 2084 LEU273 CB 7.669−20.839 −8.137 2085 LEU273 CG 8.109 −22.294 −8.354 2086 LEU273 CD1 7.859−22.713 −9.808 2087 LEU273 CD2 9.577 −22.515 −8.047 2088 VAL274 N 8.026−17.773 −7.475 2089 VAL274 CA 7.432 −16.492 −7.872 2090 VAL274 C 7.657−16.369 −9.387 2091 VAL274 O 8.406 −17.132 −9.998 2092 VAL274 CB 8.092−15.34 −7.081 2093 VAL274 CG1 7.194 −14.107 −6.99 2094 VAL274 CG2 9.471−14.952 −7.61 2095 LYS275 N 7.044 −15.285 −9.983 2096 LYS275 CA 7.464−14.86 −11.304 2097 LYS275 C 7.322 −13.332 −11.429 2098 LYS275 O 6.235−12.753 −11.4 2099 LYS275 CB 6.633 −15.505 −12.406 2100 LYS275 CG 7.255−15.212 −13.764 2101 LYS275 CD 6.224 −15.112 −14.878 2102 LYS275 CE6.828 −14.538 −16.146 2103 LYS275 NZ 7.123 −13.098 −16.004 2104 LEU276 N8.531 −12.693 −11.629 2105 LEU276 CA 8.634 −11.276 −11.95 2106 LEU276 C8.444 −11.093 −13.474 2107 LEU276 O 8.563 −12.011 −14.289 2108 LEU276 CB10.025 −10.736 −11.562 2109 LEU276 CG 10.275 −10.475 −10.064 2110 LEU276CD1 9.462 −9.29 −9.559 2111 LEU276 CD2 10.031 −11.687 −9.173 2112 ARG277N 8.207 −9.79 −13.855 2113 ARG277 CA 8.528 −9.281 −15.18 2114 ARG277 C9.232 −7.926 −14.936 2115 ARG277 O 8.825 −7.1 −14.115 2116 ARG277 CB7.289 −9.089 −16.071 2117 ARG277 CG 7.647 −8.941 −17.558 2118 ARG277 CD6.742 −7.976 −18.331 2119 ARG277 NE 7.367 −7.619 −19.624 2120 ARG277 CZ7.335 −6.381 −20.171 2121 ARG277 NH1 6.551 −5.395 −19.697 2122 ARG277NH2 8.113 −6.055 −21.217 2123 ASN278 N 10.335 −7.719 −15.749 2124 ASN278CA 10.937 −6.394 −15.894 2125 ASN278 C 10.093 −5.697 −16.971 2126 ASN278O 9.954 −6.213 −18.087 2127 ASN278 CB 12.38 −6.516 −16.379 2128 ASN278CG 12.947 −5.17 −16.753 2129 ASN278 OD1 13.081 −4.819 −17.922 2130ASN278 ND2 13.226 −4.345 −15.705 2131 PRO279 N 9.541 −4.466 −16.682 2132PRO279 CA 8.574 −3.868 −17.602 2133 PRO279 C 9.153 −3.581 −18.99 2134PRO279 O 8.444 −3.571 −19.998 2135 PRO279 CB 8.162 −2.571 −16.921 2136PRO279 CG 8.254 −2.911 −15.445 2137 PRO279 CD 9.464 −3.83 −15.371 2138TRP280 N 10.496 −3.269 −18.983 2139 TRP280 CA 11.252 −2.927 −20.181 2140TRP280 C 11.773 −4.151 −20.963 2141 TRP280 O 12.441 −4.014 −21.997 2142TRP280 CB 12.425 −1.995 −19.834 2143 TRP280 CG 12.051 −0.559 −19.6342144 TRP280 CD1 11.011 0.111 −20.238 2145 TRP280 CD2 12.756 0.413−18.852 2146 TRP280 NE1 11.062 1.433 −19.885 2147 TRP280 CE2 12.0881.633 −18.993 2148 TRP280 CE3 13.913 0.373 −18.044 2149 TRP280 CZ212.486 2.793 −18.323 2150 TRP280 CZ3 14.341 1.535 −17.391 2151 TRP280CH2 13.626 2.723 −17.518 2152 GLY281 N 11.411 −5.382 −20.457 2153 GLY281CA 11.425 −6.582 −21.268 2154 GLY281 C 12.714 −7.392 −21.278 2155 GLY281O 12.875 −8.304 −22.095 2156 LYS282 N 13.616 −7.04 −20.301 2157 LYS282CA 14.908 −7.695 −20.115 2158 LYS282 C 14.766 −8.868 −19.119 2159 LYS282O 13.791 −9.007 −18.381 2160 LYS282 CB 15.966 −6.676 −19.648 2161 LYS282CG 16.466 −5.772 −20.791 2162 LYS282 CD 15.559 −4.566 −21.044 2163LYS282 CE 15.714 −4.002 −22.451 2164 LYS282 NZ 14.659 −2.99 −22.664 2165VAL283 N 15.825 −9.754 −19.149 2166 VAL283 CA 15.911 −10.959 −18.3242167 VAL283 C 17.177 −10.793 −17.449 2168 VAL283 O 17.109 −10.559−16.244 2169 VAL283 CB 15.921 −12.225 −19.213 2170 VAL283 CG1 15.998−13.494 −18.366 2171 VAL283 CG2 14.674 −12.299 −20.104 2172 GLU284 N18.379 −10.886 −18.141 2173 GLU284 CA 19.583 −10.233 −17.632 2174 GLU284C 20.03 −10.638 −16.205 2175 GLU284 O 20.417 −9.814 −15.373 2176 GLU284CB 19.515 −8.702 −17.837 2177 GLU284 CG 19.846 −8.253 −19.27 2178 GLU284CD 18.886 −8.593 −20.412 2179 GLU284 OE1 18.035 −9.506 −20.171 2180GLU284 OE2 19.025 −7.906 −21.459 2181 TRP285 N 20.155 −12.004 −15.9852182 TRP285 CA 20.932 −12.497 −14.842 2183 TRP285 C 21.761 −13.735−15.237 2184 TRP285 O 21.657 −14.301 −16.323 2185 TRP285 CB 20.117−12.623 −13.545 2186 TRP285 CG 19.575 −13.984 −13.239 2187 TRP285 CD120.1 −14.89 −12.334 2188 TRP285 CD2 18.396 −14.581 −13.783 2189 TRP285NE1 19.303 −16.003 −12.321 2190 TRP285 CE2 18.241 −15.827 −13.175 2191TRP285 CE3 17.431 −14.167 −14.72 2192 TRP285 CZ2 17.159 −16.669 −13.4412193 TRP285 CZ3 16.37 −15.024 −15.03 2194 TRP285 CH2 16.238 −16.257−14.401 2195 LYS286 N 22.694 −14.106 −14.287 2196 LYS286 CA 23.764−15.067 −14.54 2197 LYS286 C 23.324 −16.517 −14.251 2198 LYS286 O 23.999−17.284 −13.562 2199 LYS286 CB 24.995 −14.701 −13.688 2200 LYS286 CG25.529 −13.29 −13.965 2201 LYS286 CD 26.7 −12.953 −13.039 2202 LYS286 CE27.191 −11.531 −13.271 2203 LYS286 NZ 28.293 −11.237 −12.323 2204 GLY287N 22.185 −16.93 −14.91 2205 GLY287 CA 21.742 −18.311 −14.832 2206 GLY287C 20.289 −18.461 −15.26 2207 GLY287 O 19.729 −17.631 −15.971 2208 ASP288N 19.681 −19.612 −14.781 2209 ASP288 CA 18.234 −19.828 −14.932 2210ASP288 C 17.649 −20.449 −13.637 2211 ASP288 O 16.602 −21.088 −13.6022212 ASP288 CB 17.889 −20.638 −16.168 2213 ASP288 CG 16.404 −20.537−16.444 2214 ASP288 OD1 15.815 −21.581 −16.865 2215 ASP288 OD2 15.809−19.437 −16.223 2216 TRP289 N 18.357 −20.074 −12.511 2217 TRP289 CA17.877 −20.166 −11.129 2218 TRP289 C 19.03 −19.498 −10.338 2219 TRP289 O19.797 −18.685 −10.878 2220 TRP289 CB 17.536 −21.597 −10.681 2221 TRP289CG 16.279 −21.693 −9.854 2222 TRP289 CD1 15.892 −20.886 −8.8 2223 TRP289CD2 15.271 −22.707 −9.975 2224 TRP289 NE1 14.706 −21.358 −8.3 2225TRP289 CE2 14.29 −22.451 −9.016 2226 TRP289 CE3 15.125 −23.852 −10.782227 TRP289 CZ2 13.16 −23.26 −8.852 2228 TRP289 CZ3 14.018 −24.69−10.599 2229 TRP289 CH2 13.041 −24.39 −9.659 2230 SER290 N 19.145−19.799 −9.004 2231 SER290 CA 20.299 −19.398 −8.195 2232 SER290 C 20.188−20.151 −6.87 2233 SER290 O 19.141 −20.146 −6.229 2234 SER290 CB 20.369−17.892 −7.938 2235 SER290 OG 20.955 −17.227 −9.058 2236 ASP291 N 21.338−20.833 −6.53 2237 ASP291 CA 21.456 −21.969 −5.601 2238 ASP291 C 22.001−23.178 −6.401 2239 ASP291 O 22.889 −23.913 −5.967 2240 ASP291 CB 20.238−22.329 −4.751 2241 ASP291 CG 19.008 −22.859 −5.469 2242 ASP291 OD117.956 −22.999 −4.741 2243 ASP291 OD2 19.125 −23.137 −6.697 2244 SER292N 21.344 −23.438 −7.59 2245 SER292 CA 21.699 −24.591 −8.422 2246 SER292C 20.931 −24.457 −9.74 2247 SER292 O 19.801 −24.911 −9.91 2248 SER292 CB21.375 −25.923 −7.735 2249 SER292 OG 22.418 −26.301 −6.832 2250 SER293 N21.606 −23.736 −10.724 2251 SER293 CA 20.846 −23.111 −11.818 2252 SER293C 20.023 −24.1 −12.653 2253 SER293 O 19.033 −23.732 −13.285 2254 SER293CB 21.757 −22.335 −12.787 2255 SER293 OG 21.239 −21.017 −13.017 2256SER294 N 20.552 −25.369 −12.751 2257 SER294 CA 19.9 −26.41 −13.529 2258SER294 C 18.95 −27.304 −12.721 2259 SER294 O 18.437 −28.308 −13.229 2260SER294 CB 20.925 −27.265 −14.275 2261 SER294 OG 21.73 −27.996 −13.3532262 LYS295 N 18.479 −26.832 −11.502 2263 LYS295 CA 17.627 −27.699−10.671 2264 LYS295 C 16.164 −27.847 −11.19 2265 LYS295 O 15.295 −28.434−10.547 2266 LYS295 CB 17.676 −27.362 −9.169 2267 LYS295 CG 16.925−26.088 −8.807 2268 LYS295 CD 16.949 −25.757 −7.309 2269 LYS295 CE16.153 −24.479 −7.118 2270 LYS295 NZ 16.053 −24.049 −5.734 2271 TRP296 N15.972 −27.431 −12.491 2272 TRP296 CA 14.872 −27.88 −13.336 2273 TRP296C 15.035 −29.371 −13.716 2274 TRP296 O 14.133 −29.981 −14.29 2275 TRP296CB 14.8 −27.078 −14.65 2276 TRP296 CG 14.669 −25.595 −14.468 2277 TRP296CD1 15.671 −24.659 −14.644 2278 TRP296 CD2 13.501 −24.875 −14.057 2279TRP296 NE1 15.173 −23.419 −14.334 2280 TRP296 CE2 13.856 −23.529 −13.952281 TRP296 CE3 12.188 −25.254 −13.707 2282 TRP296 CZ2 12.979 −22.546−13.477 2283 TRP296 CZ3 11.306 −24.286 −13.215 2284 TRP296 CH2 11.7−22.956 −13.095 2285 GLU297 N 16.247 −29.961 −13.388 2286 GLU297 CA16.539 −31.354 −13.743 2287 GLU297 C 15.677 −32.306 −12.884 2288 GLU297O 15.277 −33.393 −13.297 2289 GLU297 CB 18.004 −31.703 −13.427 2290GLU297 CG 19.007 −31.221 −14.472 2291 GLU297 CD 20.397 −31.304 −13.8312292 GLU297 OE1 20.987 −30.192 −13.67 2293 GLU297 OE2 20.785 −32.465−13.524 2294 LEU298 N 15.589 −31.926 −11.556 2295 LEU298 CA 14.981−32.77 −10.54 2296 LEU298 C 13.519 −32.377 −10.28 2297 LEU298 O 13.091−31.231 −10.421 2298 LEU298 CB 15.78 −32.784 −9.222 2299 LEU298 CG16.265 −31.396 −8.743 2300 LEU298 CD1 15.964 −31.174 −7.264 2301 LEU298CD2 17.769 −31.245 −8.989 2302 LEU299 N 12.717 −33.443 −9.879 2303LEU299 CA 11.365 −33.264 −9.327 2304 LEU299 C 10.5 −32.443 −10.305 2305LEU299 O 9.686 −31.581 −9.965 2306 LEU299 CB 11.374 −32.701 −7.897 2307LEU299 CG 11.447 −33.753 −6.767 2308 LEU299 CD1 10.173 −34.597 −6.6792309 LEU299 CD2 12.675 −34.655 −6.851 2310 SER300 N 10.666 −32.827−11.626 2311 SER300 CA 10.223 −31.96 −12.711 2312 SER300 C 9.831 −32.797−13.923 2313 SER300 O 10.551 −32.865 −14.918 2314 SER300 CB 11.302−30.919 −13.047 2315 SER300 OG 11.474 −30.005 −11.938 2316 PRO301 N8.614 −33.454 −13.838 2317 PRO301 CA 8.076 −34.181 −14.985 2318 PRO301 C7.562 −33.163 −16.023 2319 PRO301 O 7.523 −31.942 −15.833 2320 PRO301 CB6.982 −35.058 −14.38 2321 PRO301 CG 6.479 −34.227 −13.206 2322 PRO301 CD7.752 −33.593 −12.669 2323 LYS302 N 7.147 −33.74 −17.215 2324 LYS302 CA6.877 −32.868 −18.363 2325 LYS302 C 5.645 −31.983 −18.078 2326 LYS302 O5.484 −30.869 −18.57 2327 LYS302 CB 6.672 −33.716 −19.625 2328 LYS302 CG7.287 −33.047 −20.856 2329 LYS302 CD 7.05 −33.874 −22.119 2330 LYS302 CE7.704 −33.225 −23.329 2331 LYS302 NZ 7.445 −34.06 −24.526 2332 GLU303 N4.738 −32.591 −17.247 2333 GLU303 CA 3.395 −32.129 −16.953 2334 GLU303 C3.494 −30.92 −16.005 2335 GLU303 O 2.6 −30.081 −15.915 2336 GLU303 CB2.6 −33.255 −16.265 2337 GLU303 CG 2.426 −34.527 −17.11 2338 GLU303 CD3.625 −35.48 −17.279 2339 GLU303 OE1 4.772 −34.963 −17.076 2340 GLU303OE2 3.337 −36.652 −17.636 2341 LYS304 N 4.62 −30.928 −15.189 2342 LYS304CA 4.963 −29.757 −14.397 2343 LYS304 C 5.639 −28.73 −15.32 2344 LYS304 O5.311 −27.541 −15.299 2345 LYS304 CB 5.895 −30.123 −13.229 2346 LYS304CG 5.971 −29.01 −12.169 2347 LYS304 CD 7.075 −29.248 −11.128 2348 LYS304CE 8.442 −28.814 −11.643 2349 LYS304 NZ 9.501 −29.237 −10.709 2350ILE305 N 6.707 −29.199 −16.076 2351 ILE305 CA 7.642 −28.242 −16.673 2352ILE305 C 7.027 −27.482 −17.864 2353 ILE305 O 7.481 −26.394 −18.219 2354ILE305 CB 9.006 −28.894 −17.018 2355 ILE305 CG1 10.144 −27.856 −16.8922356 ILE305 CG2 9.013 −29.558 −18.397 2357 ILE305 CD1 11.534 −28.452−17.059 2358 LEU306 N 5.999 −28.115 −18.541 2359 LEU306 CA 5.39 −27.484−19.715 2360 LEU306 C 4.473 −26.329 −19.282 2361 LEU306 O 4.199 −25.396−20.035 2362 LEU306 CB 4.555 −28.471 −20.543 2363 LEU306 CG 5.396−29.398 −21.441 2364 LEU306 CD1 4.513 −30.523 −21.988 2365 LEU306 CD26.043 −28.646 −22.606 2366 LEU307 N 3.896 −26.466 −18.035 2367 LEU307 CA3.011 −25.447 −17.465 2368 LEU307 C 3.873 −24.476 −16.624 2369 LEU307 O3.586 −24.139 −15.471 2370 LEU307 CB 1.901 −26.13 −16.649 2371 LEU307 CG0.531 −25.423 −16.742 2372 LEU307 CD1 −0.532 −26.257 −16.022 2373 LEU307CD2 0.541 −24.005 −16.174 2374 LEU308 N 4.949 −23.941 −17.302 2375LEU308 CA 6.035 −23.215 −16.647 2376 LEU308 C 6.876 −22.584 −17.768 2377LEU308 O 7.035 −23.144 −18.85 2378 LEU308 CB 6.897 −24.178 −15.807 2379LEU308 CG 6.948 −23.815 −14.312 2380 LEU308 CD1 7.362 −25.033 −13.4862381 LEU308 CD2 7.91 −22.662 −14.059 2382 ARG309 N 7.444 −21.368 −17.442383 ARG309 CA 8.209 −20.556 −18.396 2384 ARG309 C 7.215 −20.097 −19.4992385 ARG309 O 7.211 −20.57 −20.633 2386 ARG309 CB 9.475 −21.225 −18.9562387 ARG309 CG 10.305 −22.098 −18.003 2388 ARG309 CD 10.578 −21.562−16.604 2389 ARG309 NE 11.464 −20.401 −16.533 2390 ARG309 CZ 12.822−20.438 −16.568 2391 ARG309 NH1 13.482 −21.55 −16.944 2392 ARG309 NH213.489 −19.33 −16.193 2393 LYS310 N 6.259 −19.194 −19.052 2394 LYS310 CA4.947 −19.081 −19.688 2395 LYS310 C 4.447 −17.723 −20.214 2396 LYS310 O3.452 −17.693 −20.947 2397 LYS310 CB 3.872 −19.736 −18.785 2398 LYS310CG 3.685 −19.061 −17.42 2399 LYS310 CD 2.648 −17.934 −17.42 2400 LYS310CE 2.777 −17.078 −16.165 2401 LYS310 NZ 2.02 −15.832 −16.318 2402 ASP311N 5.044 −16.569 −19.735 2403 ASP311 CA 4.74 −15.299 −20.408 2404 ASP311C 5.793 −15.231 −21.565 2405 ASP311 O 6.162 −16.217 −22.208 2406 ASP311CB 4.787 −14.092 −19.465 2407 ASP311 CG 3.902 −13.966 −18.244 2408ASP311 OD1 4.261 −13.082 −17.402 2409 ASP311 OD2 2.909 −14.74 −18.1222410 ASN312 N 6.275 −13.977 −21.862 2411 ASN312 CA 7.564 −13.796 −22.5262412 ASN312 C 8.052 −12.481 −21.892 2413 ASN312 O 7.388 −11.907 −21.0232414 ASN312 CB 7.365 −13.694 −24.032 2415 ASN312 CG 8.686 −13.81 −24.7522416 ASN312 OD1 9.415 −12.829 −24.929 2417 ASN312 ND2 9.044 −15.07−25.117 2418 ASP313 N 9.252 −11.973 −22.353 2419 ASP313 CA 9.486 −10.538−22.209 2420 ASP313 C 9.702 −10.172 −20.729 2421 ASP313 O 9.242 −9.152−20.219 2422 ASP313 CB 8.356 −9.744 −22.863 2423 ASP313 CG 9.03 −8.547−23.469 2424 ASP313 OD1 8.905 −7.459 −22.843 2425 ASP313 OD2 9.677−8.778 −24.555 2426 GLY314 N 10.58 −11.035 −20.099 2427 GLY314 CA 10.855−10.98 −18.679 2428 GLY314 C 10.158 −12.165 −18.019 2429 GLY314 O 9.038−12.091 −17.509 2430 GLU315 N 10.88 −13.349 −18.127 2431 GLU315 CA10.289 −14.613 −17.709 2432 GLU315 C 10.687 −14.949 −16.262 2433 GLU315O 9.83 −15.216 −15.422 2434 GLU315 CB 10.576 −15.729 −18.718 2435 GLU315CG 9.7 −16.972 −18.527 2436 GLU315 CD 8.191 −16.747 −18.473 2437 GLU315OE1 7.717 −15.846 −19.212 2438 GLU315 OE2 7.517 −17.508 −17.705 2439PHE316 N 12.03 −15.012 −15.961 2440 PHE316 CA 12.47 −15.188 −14.559 2441PHE316 C 12.08 −16.622 −14.073 2442 PHE316 O 12.298 −17.599 −14.803 2443PHE316 CB 12.074 −14.002 −13.659 2444 PHE316 CG 12.736 −12.706 −14.0722445 PHE316 CD1 12.085 −11.8 −14.916 2446 PHE316 CD2 14.029 −12.408−13.619 2447 PHE316 CE1 12.726 −10.628 −15.319 2448 PHE316 CE2 14.666−11.235 −14.021 2449 PHE316 CZ 14.015 −10.347 −14.874 2450 TRP317 N11.496 −16.719 −12.823 2451 TRP317 CA 11.006 −17.943 −12.169 2452 TRP317C 12.038 −18.518 −11.196 2453 TRP317 O 12.339 −19.713 −11.19 2454 TRP317CB 10.477 −19.098 −13.034 2455 TRP317 CG 9.177 −18.858 −13.717 2456TRP317 CD1 9.014 −18.346 −14.977 2457 TRP317 CD2 7.867 −19.138 −13.2232458 TRP317 NE1 7.687 −18.372 −15.307 2459 TRP317 CE2 6.957 −18.85−14.244 2460 TRP317 CE3 7.371 −19.616 −11.994 2461 TRP317 CZ2 5.578−19.027 −14.082 2462 TRP317 CZ3 5.996 −19.79 −11.817 2463 TRP317 CH25.113 −19.497 −12.85 2464 MET318 N 12.499 −17.644 −10.234 2465 MET318 CA13.197 −18.156 −9.071 2466 MET318 C 12.185 −18.312 −7.905 2467 MET318 O10.996 −18.594 −8.078 2468 MET318 CB 14.469 −17.368 −8.77 2469 MET318 CG15.331 −17.18 −10.014 2470 MET318 SD 16.964 −16.506 −9.576 2471 MET318CE 16.727 −14.789 −10.107 2472 THR319 N 12.757 −18.296 −6.644 2473THR319 CA 12.098 −18.913 −5.49 2474 THR319 C 12.337 −18.067 −4.244 2475THR319 O 13.343 −17.371 −4.135 2476 THR319 CB 12.703 −20.312 −5.228 2477THR319 OG1 14.001 −20.424 −5.84 2478 THR319 CG2 11.866 −21.415 −5.8262479 LEU320 N 11.403 −18.247 −3.239 2480 LEU320 CA 11.304 −17.264 −2.1582481 LEU320 C 12.534 −17.265 −1.231 2482 LEU320 O 12.769 −18.16 −0.4222483 LEU320 CB 10.046 −17.551 −1.315 2484 LEU320 CG 9.838 −16.663 −0.0692485 LEU320 CD1 9.894 −15.167 −0.371 2486 LEU320 CD2 8.505 −17.02 0.5962487 GLN321 N 13.316 −16.124 −1.361 2488 GLN321 CA 14.259 −15.744 −0.3112489 GLN321 C 14.594 −14.23 −0.308 2490 GLN321 O 15.433 −13.779 0.4722491 GLN321 CB 15.548 −16.59 −0.36 2492 GLN321 CG 15.855 −17.306 0.9582493 GLN321 CD 16.904 −16.635 1.819 2494 GLN321 OE1 17.909 −17.223 2.2112495 GLN321 NE2 16.654 −15.35 2.201 2496 ASP322 N 13.848 −13.446 −1.1642497 ASP322 CA 14.248 −12.103 −1.611 2498 ASP322 C 14.249 −10.983 −0.5622499 ASP322 O 14.875 −9.931 −0.719 2500 ASP322 CB 15.535 −12.116 −2.4382501 ASP322 CG 15.34 −11.89 −3.933 2502 ASP322 OD1 14.15 −11.758 −4.352503 ASP322 OD2 16.426 −11.883 −4.604 2504 PHE323 N 13.365 −11.161 0.4842505 PHE323 CA 12.822 −10.018 1.237 2506 PHE323 C 13.889 −9.399 2.1722507 PHE323 O 13.821 −9.433 3.397 2508 PHE323 CB 11.584 −10.422 2.0542509 PHE323 CG 10.362 −10.884 1.285 2510 PHE323 CD1 9.301 −11.438 2.0222511 PHE323 CD2 10.212 −10.75 −0.103 2512 PHE323 CE1 8.125 −11.847 1.3932513 PHE323 CE2 9.041 −11.175 −0.733 2514 PHE323 CZ 7.997 −11.712 0.0162515 LYS324 N 14.906 −8.775 1.479 2516 LYS324 CA 16.059 −8.143 2.0972517 LYS324 C 15.945 −6.647 1.722 2518 LYS324 O 15.583 −5.78 2.514 2519LYS324 CB 17.384 −8.77 1.596 2520 LYS324 CG 17.441 −10.305 1.664 2521LYS324 CD 18.492 −10.874 0.693 2522 LYS324 CE 18.146 −12.301 0.28 2523LYS324 NZ 19.058 −12.75 −0.782 2524 THR325 N 16.254 −6.375 0.404 2525THR325 CA 16.294 −5.05 −0.205 2526 THR325 C 14.889 −4.694 −0.708 2527THR325 O 14.517 −5.055 −1.83 2528 THR325 CB 17.024 −3.925 0.565 2529THR325 OG1 17.428 −2.899 −0.365 2530 THR325 CG2 18.294 −4.377 1.287 2531HIS326 N 14.032 −4.082 0.182 2532 HIS326 CA 12.651 −3.745 −0.185 2533HIS326 C 12.125 −2.627 0.715 2534 HIS326 O 12.506 −2.495 1.878 2535HIS326 CB 11.71 −4.964 −0.039 2536 HIS326 CG 11.237 −5.489 −1.35 2537HIS326 ND1 12.091 −5.768 −2.384 2538 HIS326 CD2 9.99 −5.805 −1.841 2539HIS326 CE1 11.334 −6.159 −3.442 2540 HIS326 NE2 10.057 −6.237 −3.1422541 PHE327 N 11.119 −1.865 0.135 2542 PHE327 CA 10.379 −0.87 0.91 2543PHE327 C 8.879 −1.214 0.924 2544 PHE327 O 8.273 −1.356 1.992 2545 PHE327CB 10.648 0.554 0.401 2546 PHE327 CG 9.97 1.626 1.224 2547 PHE327 CD110.239 1.762 2.594 2548 PHE327 CD2 9.058 2.506 0.626 2549 PHE327 CE19.585 2.734 3.353 2550 PHE327 CE2 8.404 3.477 1.386 2551 PHE327 CZ 8.6633.588 2.75 2552 VAL328 N 8.241 −1.278 −0.304 2553 VAL328 CA 6.78 −1.405−0.381 2554 VAL328 C 6.367 −2.087 −1.695 2555 VAL328 O 6.835 −1.75 −2.782556 VAL328 CB 6.081 −0.035 −0.188 2557 VAL328 CG1 6.279 0.921 −1.3692558 VAL328 CG2 4.586 −0.186 0.103 2559 LEU329 N 5.431 −3.088 −1.5412560 LEU329 CA 4.722 −3.699 −2.671 2561 LEU329 C 3.282 −3.186 −2.5862562 LEU329 O 2.749 −2.915 −1.507 2563 LEU329 CB 4.817 −5.23 −2.55 2564LEU329 CG 4.055 −6.055 −3.609 2565 LEU329 CD1 4.816 −7.344 −3.94 2566LEU329 CD2 2.655 −6.451 −3.126 2567 LEU330 N 2.607 −3.134 −3.785 2568LEU330 CA 1.18 −2.918 −3.839 2569 LEU330 C 0.517 −3.852 −4.863 2570LEU330 O 1.06 −4.232 −5.901 2571 LEU330 CB 0.768 −1.455 −4.071 2572LEU330 CG 1.329 −0.785 −5.344 2573 LEU330 CD1 0.345 0.266 −5.863 2574LEU330 CD2 2.677 −0.104 −5.08 2575 VAL331 N −0.782 −4.176 −4.512 2576VAL331 CA −1.727 −4.683 −5.49 2577 VAL331 C −2.826 −3.621 −5.632 2578VAL331 O −3.195 −2.922 −4.684 2579 VAL331 CB −2.31 −6.064 −5.13 2580VAL331 CG1 −1.224 −7.136 −5.153 2581 VAL331 CG2 −3.01 −6.103 −3.774 2582ILE332 N −3.381 −3.556 −6.897 2583 ILE332 CA −4.589 −2.77 −7.148 2584ILE332 C −5.721 −3.807 −7.228 2585 ILE332 O −5.873 −4.569 −8.182 2586ILE332 CB −4.423 −1.846 −8.371 2587 ILE332 CG1 −5.382 −0.638 −8.329 2588ILE332 CG2 −4.482 −2.546 −9.731 2589 ILE332 CD1 −6.855 −0.957 −8.1582590 CYS333 N −6.476 −3.899 −6.073 2591 CYS333 CA −7.589 −4.832 −5.9762592 CYS333 C −8.783 −4.185 −6.689 2593 CYS333 O −9.295 −3.132 −6.3092594 CYS333 CB −7.977 −5.116 −4.53 2595 CYS333 SG −6.842 −6.3 −3.7432596 LYS334 N −9.182 −4.88 −7.816 2597 LYS334 CA −10.111 −4.323 −8.7922598 LYS334 C −11.546 −4.279 −8.235 2599 LYS334 O −11.95 −5.048 −7.3652600 LYS334 CB −10.11 −5.186 −10.065 2601 LYS334 CG −8.863 −4.966−10.925 2602 LYS334 CD −8.707 −6.065 −11.976 2603 LYS334 CE −7.611−5.715 −12.971 2604 LYS334 NZ −7.367 −6.877 −13.845 2605 LEU335 N−12.355 −3.345 −8.864 2606 LEU335 CA −13.81 −3.365 −8.711 2607 LEU335 C−14.436 −4.308 −9.769 2608 LEU335 O −13.753 −4.987 −10.536 2609 LEU335CB −14.404 −1.947 −8.725 2610 LEU335 CG −14.005 −0.994 −9.87 2611 LEU335CD1 −14.051 −1.609 −11.257 2612 LEU335 CD2 −14.918 0.238 −9.852 2613THR336 N −15.823 −4.328 −9.782 2614 THR336 CA −16.561 −5.327 −10.5692615 THR336 C −16.279 −5.158 −12.078 2616 THR336 O −15.707 −6.075−12.687 2617 THR336 CB −18.053 −5.318 −10.19 2618 THR336 OG1 −18.175−5.305 −8.755 2619 THR336 CG2 −18.802 −6.527 −10.736 2620 PRO337 N−16.567 −3.978 −12.736 2621 PRO337 CA −16.139 −3.762 −14.13 2622 PRO337C −14.623 −3.439 −14.198 2623 PRO337 O −14.151 −2.408 −14.688 2624PRO337 CB −17.009 −2.603 −14.62 2625 PRO337 CG −17.292 −1.815 −13.3462626 PRO337 CD −17.465 −2.912 −12.306 2627 GLY338 N −13.819 −4.463−13.734 2628 GLY338 CA −12.369 −4.376 −13.653 2629 GLY338 C −11.676−4.999 −14.864 2630 GLY338 O −10.467 −4.881 −15.049 2631 LEU339 N−12.517 −5.75 −15.649 2632 LEU339 CA −12.165 −6.392 −16.909 2633 LEU339C −13.452 −6.384 −17.749 2634 LEU339 O −14.556 −6.131 −17.257 2635LEU339 CB −11.709 −7.847 −16.695 2636 LEU339 CG −10.31 −8 −16.073 2637LEU339 CD1 −10.003 −9.479 −15.823 2638 LEU339 CD2 −9.234 −7.392 −16.9712639 LEU340 N −13.287 −6.756 −19.068 2640 LEU340 CA −14.439 −6.983−19.944 2641 LEU340 C −14.996 −8.379 −19.597 2642 LEU340 O −14.778−9.384 −20.267 2643 LEU340 CB −14.015 −6.863 −21.414 2644 LEU340 CG−15.167 −6.977 −22.434 2645 LEU340 CD1 −16.231 −5.895 −22.245 2646LEU340 CD2 −14.603 −6.904 −23.856 2647 SER341 N −15.653 −8.423 −18.3752648 SER341 CA −16.715 −9.391 −18.078 2649 SER341 C −16.338 −10.882−18.154 2650 SER341 O −17.186 −11.77 −18.227 2651 SER341 CB −18.025−9.094 −18.827 2652 SER341 OG −17.834 −9.05 −20.236 2653 GLN342 N−14.988 −11.143 −17.948 2654 GLN342 CA −14.489 −12.484 −18.241 2655GLN342 C −15.049 −13.524 −17.241 2656 GLN342 O −15.217 −13.291 −16.0382657 GLN342 CB −12.955 −12.548 −18.112 2658 GLN342 CG −12.195 −11.889−19.267 2659 GLN342 CD −10.69 −11.852 −19.048 2660 GLN342 OE1 −9.978−10.911 −19.398 2661 GLN342 NE2 −10.135 −12.963 −18.478 2662 GLU343 N−15.17 −14.791 −17.779 2663 GLU343 CA −15.785 −15.89 −17.033 2664 GLU343C −14.716 −16.454 −16.084 2665 GLU343 O −14.982 −16.976 −15.005 2666GLU343 CB −16.253 −17.031 −17.956 2667 GLU343 CG −17.318 −16.631 −18.9812668 GLU343 CD −16.779 −15.923 −20.232 2669 GLU343 OE1 −17.457 −16.091−21.28 2670 GLU343 OE2 −15.72 −15.245 −20.048 2671 ALA344 N −13.443−16.438 −16.632 2672 ALA344 CA −12.281 −16.858 −15.876 2673 ALA344 C−11.844 −15.727 −14.926 2674 ALA344 O −12.048 −14.533 −15.162 2675ALA344 CB −11.13 −17.223 −16.802 2676 ALA345 N −11.171 −16.198 −13.8112677 ALA345 CA −10.751 −15.378 −12.674 2678 ALA345 C −11.96 −14.979−11.811 2679 ALA345 O −13.024 −14.585 −12.3 2680 ALA345 CB −9.926 −14.16−13.056 2681 GLN346 N −11.726 −15.069 −10.444 2682 GLN346 CA −12.774−14.685 −9.503 2683 GLN346 C −12.841 −13.153 −9.405 2684 GLN346 O −11.88−12.418 −9.633 2685 GLN346 CB −12.573 −15.264 −8.103 2686 GLN346 CG−12.839 −16.767 −8.015 2687 GLN346 CD −13.021 −17.178 −6.565 2688 GLN346OE1 −13.722 −16.548 −5.776 2689 GLN346 NE2 −12.378 −18.327 −6.205 2690LYS347 N −14.088 −12.681 −9.037 2691 LYS347 CA −14.468 −11.268 −9.0842692 LYS347 C −15.517 −11.068 −7.972 2693 LYS347 O −15.975 −12.017−7.333 2694 LYS347 CB −15.011 −10.884 −10.479 2695 LYS347 CG −14.023−11.248 −11.595 2696 LYS347 CD −14.491 −10.939 −13.015 2697 LYS347 CE−13.508 −11.511 −14.04 2698 LYS347 NZ −13.675 −12.969 −14.176 2699TRP348 N −15.889 −9.76 −7.745 2700 TRP348 CA −16.666 −9.383 −6.56 2701TRP348 C −17.466 −8.095 −6.815 2702 TRP348 O −17.235 −7.335 −7.758 2703TRP348 CB −15.803 −9.263 −5.289 2704 TRP348 CG −14.525 −8.481 −5.4212705 TRP348 CD1 −14.24 −7.437 −6.28 2706 TRP348 CD2 −13.347 −8.677−4.623 2707 TRP348 NE1 −12.934 −7.071 −6.103 2708 TRP348 CE2 −12.359−7.828 −5.115 2709 TRP348 CE3 −13.023 −9.513 −3.536 2710 TRP348 CZ2−11.051 −7.818 −4.623 2711 TRP348 CZ3 −11.735 −9.471 −2.99 2712 TRP348CH2 −10.755 −8.653 −3.544 2713 THR349 N −18.461 −7.859 −5.885 2714THR349 CA −19.45 −6.792 −6.055 2715 THR349 C −18.885 −5.466 −5.521 2716THR349 O −18.407 −5.345 −4.393 2717 THR349 CB −20.729 −7.127 −5.256 2718THR349 OG1 −20.996 −8.532 −5.384 2719 THR349 CG2 −21.937 −6.338 −5.7452720 TYR350 N −19.048 −4.388 −6.376 2721 TYR350 CA −18.566 −3.041 −6.0112722 TYR350 C −19.482 −2.397 −4.937 2723 TYR350 O −20.094 −1.341 −5.1062724 TYR350 CB −18.5 −2.155 −7.273 2725 TYR350 CG −17.919 −0.767 −7.1012726 TYR350 CD1 −16.742 −0.538 −6.376 2727 TYR350 CD2 −18.552 0.325−7.718 2728 TYR350 CE1 −16.227 0.755 −6.246 2729 TYR350 CE2 −18.0411.618 −7.586 2730 TYR350 CZ −16.891 1.825 −6.839 2731 TYR350 OH −16.4343.104 −6.694 2732 THR351 N −19.482 −3.031 −3.709 2733 THR351 CA −20.34−2.593 −2.598 2734 THR351 C −19.646 −1.44 −1.834 2735 THR351 O −19.535−1.419 −0.609 2736 THR351 CB −20.687 −3.766 −1.655 2737 THR351 OG1−21.089 −4.902 −2.437 2738 THR351 CG2 −21.844 −3.438 −0.709 2739 MET352N −19.26 −0.379 −2.635 2740 MET352 CA −18.829 0.901 −2.074 2741 MET352 C−20.09 1.772 −1.929 2742 MET352 O −21.078 1.635 −2.649 2743 MET352 CB−17.791 1.551 −3 2744 MET352 CG −17.031 2.699 −2.336 2745 MET352 SD−15.698 3.301 −3.427 2746 MET352 CE −14.9 4.439 −2.265 2747 ARG353 N−20.012 2.736 −0.941 2748 ARG353 CA −21.065 3.727 −0.741 2749 ARG353 C−20.409 5.118 −0.686 2750 ARG353 O −19.193 5.265 −0.578 2751 ARG353 CB−21.866 3.44 0.534 2752 ARG353 CG −22.613 2.105 0.469 2753 ARG353 CD−23.63 1.995 1.594 2754 ARG353 NE −24.282 0.679 1.613 2755 ARG353 CZ−23.829 −0.389 2.309 2756 ARG353 NH1 −24.598 −1.488 2.418 2757 ARG353NH2 −22.633 −0.385 2.934 2758 GLU354 N −21.313 6.159 −0.787 2759 GLU354CA −20.907 7.561 −0.682 2760 GLU354 C −21.009 8.025 0.787 2761 GLU354 O−21.52 7.333 1.669 2762 GLU354 CB −21.795 8.418 −1.607 2763 GLU354 CG−21.021 8.966 −2.806 2764 GLU354 CD −19.941 9.964 −2.419 2765 GLU354 OE1−19.803 10.212 −1.187 2766 GLU354 OE2 −19.268 10.453 −3.384 2767 GLY355N −20.49 9.288 1.024 2768 GLY355 CA −20.444 9.829 2.368 2769 GLY355 C−20.044 11.301 2.472 2770 GLY355 O −19.549 11.947 1.552 2771 ARG356 N−20.278 11.835 3.732 2772 ARG356 CA −19.973 13.22 4.076 2773 ARG356 C−19.435 13.261 5.517 2774 ARG356 O −19.822 12.473 6.377 2775 ARG356 CB−21.22 14.11 4.019 2776 ARG356 CG −21.71 14.391 2.6 2777 ARG356 CD−23.015 15.181 2.591 2778 ARG356 NE −24.15 14.34 3.001 2779 ARG356 CZ−24.814 14.395 4.183 2780 ARG356 NH1 −25.871 13.564 4.361 2781 ARG356NH2 −24.496 15.216 5.191 2782 TRP357 N −18.54 14.291 5.734 2783 TRP357CA −17.903 14.556 7.028 2784 TRP357 C −17.788 16.092 7.12 2785 TRP357 O−16.762 16.695 6.804 2786 TRP357 CB −16.508 13.905 7.125 2787 TRP357 CG−16.539 12.489 7.622 2788 TRP357 CD1 −16.376 12.093 8.934 2789 TRP357CD2 −16.724 11.29 6.859 2790 TRP357 NE1 −16.508 10.732 9.009 2791 TRP357CE2 −16.721 10.216 7.753 2792 TRP357 CE3 −16.909 11.018 5.486 2793TRP357 CZ2 −16.913 8.891 7.346 2794 TRP357 CZ3 −17.116 9.701 5.064 2795TRP357 CH2 −17.119 8.656 5.983 2796 GLU358 N −18.962 16.734 7.473 2797GLU358 CA −19.026 18.171 7.728 2798 GLU358 C −18.992 18.415 9.253 2799GLU358 O −19.324 17.567 10.083 2800 GLU358 CB −20.231 18.902 7.101 2801GLU358 CG −21.012 18.159 6.019 2802 GLU358 CD −22.066 17.165 6.495 2803GLU358 OE1 −22.758 16.626 5.577 2804 GLU358 OE2 −22.149 16.952 7.7472805 LYS359 N −18.551 19.678 9.621 2806 LYS359 CA −18.467 20.053 11.0282807 LYS359 C −19.789 20.701 11.473 2808 LYS359 O −20.366 21.552 10.7922809 LYS359 CB −17.316 21.036 11.288 2810 LYS359 CG −15.946 20.357 11.182811 LYS359 CD −14.806 21.321 11.516 2812 LYS359 CE −13.46 20.611 11.4542813 LYS359 NZ −12.388 21.56 11.839 2814 ARG360 N −20.185 20.303 12.742815 ARG360 CA −21.453 20.645 13.397 2816 ARG360 C −22.571 19.638 13.0492817 ARG360 O −23.753 19.862 13.306 2818 ARG360 CB −21.969 22.07 13.1562819 ARG360 CG −20.949 23.176 13.424 2820 ARG360 CD −21.441 24.49 12.832821 ARG360 NE −20.344 25.336 12.347 2822 ARG360 CZ −19.678 25.13511.181 2823 ARG360 NH1 −18.825 26.093 10.748 2824 ARG360 NH2 −19.82724.028 10.422 2825 SER361 N −22.132 18.433 12.534 2826 SER361 CA −23.09217.492 11.962 2827 SER361 C −22.527 16.07 12.006 2828 SER361 O −23.05215.2 12.699 2829 SER361 CB −23.45 17.878 10.521 2830 SER361 OG −22.24418.109 9.793 2831 THR362 N −21.427 15.849 11.197 2832 THR362 CA −20.96714.494 10.88 2833 THR362 C −19.496 14.261 11.248 2834 THR362 O −19.0713.144 11.557 2835 THR362 CB −21.194 14.156 9.395 2836 THR362 OG1 −20.7415.237 8.575 2837 THR362 CG2 −22.654 13.848 9.079 2838 ALA363 N −18.64215.337 11.141 2839 ALA363 CA −17.212 15.227 11.413 2840 ALA363 C −1715.136 12.933 2841 ALA363 O −16.552 16.058 13.607 2842 ALA363 CB −16.43516.394 10.824 2843 GLY364 N −17.37 13.903 13.457 2844 GLY364 CA −17.58513.743 14.883 2845 GLY364 C −16.35 13.324 15.673 2846 GLY364 O −16.39813.133 16.887 2847 GLY365 N −15.212 13.151 14.922 2848 GLY365 CA −13.95212.741 15.498 2849 GLY365 C −13.734 11.229 15.406 2850 GLY365 O −14.52710.457 14.872 2851 GLN366 N −12.522 10.847 15.957 2852 GLN366 CA −12.0759.46 16.036 2853 GLN366 C −12.217 9.004 17.5 2854 GLN366 O −12.316 9.79918.438 2855 GLN366 CB −10.612 9.339 15.598 2856 GLN366 CG −10.4 9.74414.14 2857 GLN366 CD −8.968 9.503 13.72 2858 GLN366 OE1 −8.143 10.40213.608 2859 GLN366 NE2 −8.653 8.196 13.472 2860 ARG367 N −12.109 7.63417.701 2861 ARG367 CA −12.65 7.006 18.916 2862 ARG367 C −11.849 7.19720.228 2863 ARG367 O −11.982 6.425 21.184 2864 ARG367 CB −12.905 5.49818.688 2865 ARG367 CG −11.635 4.631 18.755 2866 ARG367 CD −11.779 3.4819.753 2867 ARG367 NE −10.476 3.073 20.296 2868 ARG367 CZ −9.887 3.66521.366 2869 ARG367 NH1 −10.429 4.7 22.025 2870 ARG367 NH2 −8.69 3.22621.806 2871 GLN368 N −11.05 8.313 20.291 2872 GLN368 CA −10.37 8.73221.509 2873 GLN368 C −10.05 10.237 21.513 2874 GLN368 O −9.328 10.74322.375 2875 GLN368 CB −9.092 7.906 21.735 2876 GLN368 CG −8.796 7.68823.221 2877 GLN368 CD −7.888 6.49 23.4 2878 GLN368 OE1 −8.313 5.33423.354 2879 GLN368 NE2 −6.565 6.78 23.545 2880 LEU369 N −10.702 11.00720.568 2881 LEU369 CA −10.55 12.461 20.559 2882 LEU369 C −11.611 13.03621.509 2883 LEU369 O −12.578 13.703 21.135 2884 LEU369 CB −10.684 13.04419.146 2885 LEU369 CG −9.617 12.561 18.142 2886 LEU369 CD1 −9.783 13.30116.811 2887 LEU369 CD2 −8.184 12.745 18.645 2888 LEU370 N −11.352 12.81422.853 2889 LEU370 CA −12.372 12.994 23.897 2890 LEU370 C −12.689 14.47824.253 2891 LEU370 O −13.159 14.812 25.339 2892 LEU370 CB −12.003 12.22725.183 2893 LEU370 CG −11.831 10.701 25.034 2894 LEU370 CD1 −11.4710.087 26.39 2895 LEU370 CD2 −13.076 10.017 24.47 2896 GLN371 N −12.5815.354 23.193 2897 GLN371 CA −13.322 16.61 23.121 2898 GLN371 C −14.71116.324 22.524 2899 GLN371 O −15.701 16.945 22.907 2900 GLN371 CB −12.53617.591 22.228 2901 GLN371 CG −13.169 18.976 22.049 2902 GLN371 CD−14.262 19.089 20.995 2903 GLN371 OE1 −15.348 19.606 21.238 2904 GLN371NE2 −13.921 18.669 19.739 2905 ASP372 N −14.704 15.511 21.402 2906ASP372 CA −15.799 15.527 20.44 2907 ASP372 C −16.76 14.328 20.615 2908ASP372 O −16.66 13.492 21.511 2909 ASP372 CB −15.203 15.594 19.035 2910ASP372 CG −16.111 16.58 18.31 2911 ASP372 OD1 −17.287 16.145 18.107 2912ASP372 OD2 −15.588 17.709 18.076 2913 THR373 N −17.792 14.315 19.6882914 THR373 CA −18.977 13.493 19.86 2915 THR373 C −18.777 12.024 19.4822916 THR373 O −19.582 11.178 19.884 2917 THR373 CB −20.195 14.049 19.0852918 THR373 OG1 −21.431 13.373 19.431 2919 THR373 CG2 −20.056 13.99717.571 2920 PHE374 N −17.84 11.738 18.522 2921 PHE374 CA −17.508 10.39218.051 2922 PHE374 C −18.642 9.76 17.217 2923 PHE374 O −18.488 9.45316.032 2924 PHE374 CB −17.011 9.462 19.177 2925 PHE374 CG −16.836 8.01718.764 2926 PHE374 CD1 −16.167 7.669 17.583 2927 PHE374 CD2 −17.3756.996 19.561 2928 PHE374 CE1 −16.096 6.336 17.182 2929 PHE374 CE2−17.284 5.66 19.166 2930 PHE374 CZ −16.653 5.331 17.97 2931 TRP375 N−19.817 9.514 17.898 2932 TRP375 CA −20.82 8.557 17.424 2933 TRP375 C−21.755 9.084 16.323 2934 TRP375 O −22.61 8.367 15.804 2935 TRP375 CB−21.629 7.912 18.564 2936 TRP375 CG −22.331 8.872 19.483 2937 TRP375 CD1−21.851 9.306 20.704 2938 TRP375 CD2 −23.628 9.464 19.323 2939 TRP375NE1 −22.769 10.151 21.262 2940 TRP375 CE2 −23.867 10.263 20.444 2941TRP375 CE3 −24.638 9.38 18.341 2942 TRP375 CZ2 −25.054 10.984 20.6272943 TRP375 CZ3 −25.831 10.092 18.509 2944 TRP375 CH2 −26.035 10.88119.638 2945 LYS376 N −21.516 10.383 15.928 2946 LYS376 CA −22.219 11.01114.814 2947 LYS376 C −21.41 10.936 13.501 2948 LYS376 O −21.746 11.55712.494 2949 LYS376 CB −22.584 12.47 15.123 2950 LYS376 CG −23.353 12.62516.44 2951 LYS376 CD −23.905 14.043 16.608 2952 LYS376 CE −24.52 14.26917.983 2953 LYS376 NZ −23.482 14.625 18.973 2954 ASN377 N −20.364 10.02713.505 2955 ASN377 CA −19.79 9.578 12.237 2956 ASN377 C −20.838 8.65811.589 2957 ASN377 O −21.536 7.917 12.289 2958 ASN377 CB −18.536 8.73312.44 2959 ASN377 CG −17.282 9.556 12.579 2960 ASN377 OD1 −16.574 9.83511.614 2961 ASN377 ND2 −16.991 9.935 13.859 2962 PRO378 N −20.903 8.62910.213 2963 PRO378 CA −21.896 7.798 9.54 2964 PRO378 C −21.427 6.3349.541 2965 PRO378 O −20.324 5.978 9.129 2966 PRO378 CB −21.965 8.3588.118 2967 PRO378 CG −20.59 8.984 7.909 2968 PRO378 CD −20.246 9.5369.283 2969 GLN379 N −22.355 5.455 10.063 2970 GLN379 CA −22.13 4.0210.099 2971 GLN379 C −22.575 3.441 8.742 2972 GLN379 O −23.457 3.9558.053 2973 GLN379 CB −22.913 3.342 11.236 2974 GLN379 CG −22.593 3.90512.627 2975 GLN379 CD −23.27 5.236 12.913 2976 GLN379 OE1 −24.062 5.77412.145 2977 GLN379 NE2 −22.924 5.779 14.122 2978 PHE380 N −21.936 2.2658.383 2979 PHE380 CA −22.185 1.634 7.09 2980 PHE380 C −22.63 0.18 7.2952981 PHE380 O −22.198 −0.546 8.188 2982 PHE380 CB −20.949 1.658 6.1812983 PHE380 CG −20.569 3.043 5.716 2984 PHE380 CD1 −19.357 3.616 6.1222985 PHE380 CD2 −21.416 3.779 4.873 2986 PHE380 CE1 −19.001 4.896 5.6932987 PHE380 CE2 −21.056 5.057 4.443 2988 PHE380 CZ −19.848 5.615 4.8532989 LEU381 N −23.534 −0.249 6.338 2990 LEU381 CA −24.25 −1.514 6.462991 LEU381 C −23.534 −2.548 5.567 2992 LEU381 O −23.838 −2.741 4.3882993 LEU381 CB −25.714 −1.298 6.037 2994 LEU381 CG −26.653 −2.459 6.4142995 LEU381 CD1 −26.929 −2.494 7.92 2996 LEU381 CD2 −27.978 −2.326 5.662997 LEU382 N −22.46 −3.169 6.173 2998 LEU382 CA −21.835 −4.382 5.632999 LEU382 C −22.284 −5.561 6.522 3000 LEU382 O −22.899 −5.375 7.5773001 LEU382 CB −20.31 −4.248 5.591 3002 LEU382 CG −19.805 −3.112 4.6753003 LEU382 CD1 −18.293 −2.95 4.834 3004 LEU382 CD2 −20.143 −3.357 3.2033005 SER383 N −21.988 −6.824 6.048 3006 SER383 CA −22.588 −8.052 6.6173007 SER383 C −21.629 −9.25 6.398 3008 SER383 O −20.671 −9.146 5.6233009 SER383 CB −23.949 −8.315 5.955 3010 SER383 OG −24.973 −7.4 6.3963011 VAL384 N −21.83 −10.37 7.195 3012 VAL384 CA −21.372 −11.751 6.8423013 VAL384 C −22.079 −12.761 7.821 3014 VAL384 O −22.425 −12.38 8.9423015 VAL384 CB −19.837 −11.888 6.772 3016 VAL384 CG1 −19.229 −12.2148.117 3017 VAL384 CG2 −19.373 −12.917 5.744 3018 TRP385 N −22.278 −14.067.353 3019 TRP385 CA −22.723 −15.189 8.229 3020 TRP385 C −21.53 −16.1498.431 3021 TRP385 O −20.479 −15.995 7.81 3022 TRP385 CB −24.005 −15.937.763 3023 TRP385 CG −23.947 −16.867 6.581 3024 TRP385 CD1 −23.168−18.003 6.451 3025 TRP385 CD2 −24.804 −16.854 5.419 3026 TRP385 NE1−23.438 −18.598 5.247 3027 TRP385 CE2 −24.405 −17.891 4.578 3028 TRP385CE3 −25.913 −16.085 5.006 3029 TRP385 CZ2 −24.963 −18.119 3.313 3030TRP385 CZ3 −26.5 −16.301 3.754 3031 TRP385 CH2 −26.014 −17.294 2.91 3032ARG386 N −21.73 −17.184 9.335 3033 ARG386 CA −20.636 −18.069 9.763 3034ARG386 C −21.064 −19.578 9.786 3035 ARG386 O −21.971 −19.986 10.516 3036ARG386 CB −20.162 −17.568 11.128 3037 ARG386 CG −19.024 −18.381 11.7253038 ARG386 CD −18.422 −17.636 12.911 3039 ARG386 NE −17.243 −16.86212.502 3040 ARG386 CZ −17.023 −15.559 12.755 3041 ARG386 NH1 −15.887−14.992 12.315 3042 ARG386 NH2 −17.891 −14.792 13.442 3043 PRO387 N−20.383 −20.443 8.926 3044 PRO387 CA −20.892 −21.788 8.578 3045 PRO387 C−20.298 −22.971 9.395 3046 PRO387 O −19.435 −22.831 10.267 3047 PRO387CB −20.461 −21.931 7.113 3048 PRO387 CG −19.113 −21.221 7.091 3049PRO387 CD −19.383 −20.011 7.958 3050 GLU388 N −20.764 −24.221 9.005 3051GLU388 CA −20.389 −25.505 9.623 3052 GLU388 C −19.082 −26.127 9.012 3053GLU388 O −18.926 −27.349 8.891 3054 GLU388 CB −21.524 −26.554 9.497 3055GLU388 CG −22.909 −26.148 10.003 3056 GLU388 CD −23.108 −26.087 11.5153057 GLU388 OE1 −22.765 −27.093 12.197 3058 GLU388 OE2 −23.571 −24.97911.934 3059 GLU389 N −18.033 −25.254 8.789 3060 GLU389 CA −16.664−25.697 8.397 3061 GLU389 C −15.987 −26.349 9.647 3062 GLU389 O −16.605−26.566 10.696 3063 GLU389 CB −15.93 −24.498 7.763 3064 GLU389 CG−14.713 −24.819 6.884 3065 GLU389 CD −13.399 −24.46 7.563 3066 GLU389OE1 −12.701 −23.533 7.064 3067 GLU389 OE2 −13.124 −25.121 8.61 3068GLY390 N −14.681 −26.751 9.516 3069 GLY390 CA −14.027 −27.553 10.53 3070GLY390 C −13.423 −26.714 11.662 3071 GLY390 O −14.015 −25.771 12.1953072 ARG391 N −12.162 −27.162 12.036 3073 ARG391 CA −11.427 −26.62813.18 3074 ARG391 C −10.954 −25.21 12.832 3075 ARG391 O −10.327 −24.92711.809 3076 ARG391 CB −10.224 −27.531 13.497 3077 ARG391 CG −9.386−27.126 14.715 3078 ARG391 CD −10.134 −27.236 16.041 3079 ARG391 NE−9.223 −26.995 17.173 3080 ARG391 CZ −9.596 −27.072 18.477 3081 ARG391NH1 −8.666 −26.907 19.442 3082 ARG391 NH2 −10.872 −27.303 18.843 3083ARG392 N −11.331 −24.267 13.785 3084 ARG392 CA −11.305 −22.854 13.4363085 ARG392 C −10.846 −21.965 14.609 3086 ARG392 O −11.058 −20.75314.675 3087 ARG392 CB −12.647 −22.412 12.835 3088 ARG392 CG −12.793−22.794 11.36 3089 ARG392 CD −14.19 −22.585 10.807 3090 ARG392 NE−15.126 −23.597 11.318 3091 ARG392 CZ −16.472 −23.486 11.233 3092 ARG392NH1 −17.268 −24.443 11.732 3093 ARG392 NH2 −17.067 −22.445 10.617 3094SER393 N −9.984 −22.596 15.478 3095 SER393 CA −9.333 −21.934 16.611 3096SER393 C −7.956 −21.336 16.252 3097 SER393 O −7.059 −21.28 17.093 3098SER393 CB −9.151 −22.928 17.767 3099 SER393 OG −8.707 −22.282 18.9553100 LEU394 N −7.851 −20.775 14.996 3101 LEU394 CA −6.568 −20.364 14.4043102 LEU394 C −6.794 −19.764 12.993 3103 LEU394 O −6.362 −20.323 11.9853104 LEU394 CB −5.522 −21.52 14.394 3105 LEU394 CG −5.762 −22.791 13.5393106 LEU394 CD1 −4.561 −23.734 13.699 3107 LEU394 CD2 −7.04 −23.55513.868 3108 ARG395 N −7.593 −18.622 12.89 3109 ARG395 CA −8.21 −18.311.586 3110 ARG395 C −8.701 −16.782 11.178 3111 ARG395 O −9.874 −16.52510.892 3112 ARG395 CB −9.39 −19.275 11.375 3113 ARG395 CG −9.286 −20.80611.46 3114 ARG395 CD −8.562 −21.618 10.391 3115 ARG395 NE −9.305 −21.6589.128 3116 ARG395 CZ −10.205 −22.574 8.688 3117 ARG395 NH1 −10.593−23.658 9.402 3118 ARG395 NH2 −10.761 −22.39 7.476 3119 PRO396 N −7.74−15.79 11.005 3120 PRO396 CA −7.983 −14.407 10.506 3121 PRO396 C −8.425−14.105 9.049 3122 PRO396 O −8.923 −14.954 8.312 3123 PRO396 CB −6.609−13.747 10.679 3124 PRO396 CG −5.993 −14.423 11.868 3125 PRO396 CD−6.711 −15.746 11.993 3126 CYS397 N −8.274 −12.755 8.692 3127 CYS397 CA−8.72 −12.15 7.421 3128 CYS397 C −8.328 −10.621 7.371 3129 CYS397 O−7.924 −10.054 8.394 3130 CYS397 CB −10.208 −12.41 7.221 3131 CYS397 SG−11.279 −11.876 8.582 3132 SER398 N −8.423 −9.94 6.157 3133 SER398 CA−8.182 −8.484 6.024 3134 SER398 C −9.232 −7.657 5.24 3135 SER398 O−9.673 −7.98 4.142 3136 SER398 CB −6.83 −8.139 5.399 3137 SER398 OG−6.54 −6.747 5.627 3138 VAL399 N −9.546 −6.441 5.84 3139 VAL399 CA−10.484 −5.489 5.229 3140 VAL399 C −9.877 −4.905 3.935 3141 VAL399 O−8.669 −4.858 3.714 3142 VAL399 CB −10.915 −4.399 6.256 3143 VAL399 CG1−12.368 −4.572 6.703 3144 VAL399 CG2 −10.719 −2.942 5.827 3145 LEU400 N−10.855 −4.406 3.088 3146 LEU400 CA −10.577 −3.844 1.768 3147 LEU400 C−11.708 −2.838 1.468 3148 LEU400 O −12.448 −2.909 0.491 3149 LEU400 CB−10.524 −4.964 0.726 3150 LEU400 CG −10.008 −4.493 −0.644 3151 LEU400CD1 −8.493 −4.301 −0.627 3152 LEU400 CD2 −10.408 −5.497 −1.721 3153VAL401 N −11.801 −1.835 2.407 3154 VAL401 CA −12.723 −0.702 2.301 3155VAL401 C −11.844 0.483 2.736 3156 VAL401 O −10.856 0.309 3.454 3157VAL401 CB −13.959 −0.896 3.208 3158 VAL401 CG1 −14.994 0.218 3.018 3159VAL401 CG2 −14.649 −2.245 2.967 3160 SER402 N −12.241 1.72 2.284 3161SER402 CA −11.36 2.883 2.401 3162 SER402 C −12.157 4.151 2.745 3163SER402 O −13.38 4.229 2.642 3164 SER402 CB −10.56 3.073 1.105 3165SER402 OG −11.443 3.091 −0.023 3166 LEU403 N −11.356 5.198 3.161 3167LEU403 CA −11.854 6.504 3.597 3168 LEU403 C −10.857 7.561 3.072 3169LEU403 O −10.423 8.519 3.706 3170 LEU403 CB −12.133 6.514 5.096 3171LEU403 CG −12.702 7.82 5.681 3172 LEU403 CD1 −13.726 8.51 4.783 3173LEU403 CD2 −13.325 7.552 7.053 3174 LEU404 N −10.668 7.373 1.714 3175LEU404 CA −9.941 8.264 0.812 3176 LEU404 C −10.818 9.529 0.723 3177LEU404 O −12.024 9.486 0.474 3178 LEU404 CB −9.801 7.527 −0.53 3179LEU404 CG −8.834 8.136 −1.553 3180 LEU404 CD1 −8.581 7.127 −2.68 3181LEU404 CD2 −9.37 9.432 −2.147 3182 GLN405 N −10.159 10.714 1.016 3183GLN405 CA −10.916 11.95 1.155 3184 GLN405 C −11.398 12.498 −0.194 3185GLN405 O −10.848 12.242 −1.26 3186 GLN405 CB −10.152 13.044 1.92 3187GLN405 CG −8.982 13.679 1.17 3188 GLN405 CD −8.409 14.848 1.941 3189GLN405 OE1 −8.517 16.01 1.558 3190 GLN405 NE2 −7.772 14.524 3.104 3191LYS406 N −12.457 13.383 −0.088 3192 LYS406 CA −12.778 14.302 −1.177 3193LYS406 C −12.141 15.635 −0.743 3194 LYS406 O −12.518 16.183 0.306 3195LYS406 CB −14.292 14.496 −1.302 3196 LYS406 CG −15.033 13.227 −1.7443197 LYS406 CD −16.543 13.452 −1.659 3198 LYS406 CE −17.366 12.198 −1.93199 LYS406 NZ −17.498 11.909 −3.337 3200 PRO407 N −11.126 16.161 −1.5113201 PRO407 CA −10.416 17.373 −1.111 3202 PRO407 C −11.214 18.633 −1.4953203 PRO407 O −12.201 18.619 −2.226 3204 PRO407 CB −9.092 17.307 −1.8753205 PRO407 CG −9.469 16.556 −3.147 3206 PRO407 CD −10.478 15.53 −2.6533207 ARG408 N −10.711 19.793 −0.93 3208 ARG408 CA −11.226 21.121 −1.2763209 ARG408 C −10.01 21.966 −1.669 3210 ARG408 O −8.867 21.71 −1.2733211 ARG408 CB −11.946 21.751 −0.075 3212 ARG408 CG −13.319 21.114 0.1673213 ARG408 CD −13.694 21.058 1.65 3214 ARG408 NE −14.581 19.919 1.9473215 ARG408 CZ −14.228 18.617 1.777 3216 ARG408 NH1 −12.974 18.264 1.4443217 ARG408 NH2 −15.149 17.647 1.94 3218 HIS409 N −10.29 23.114 −2.3863219 HIS409 CA −9.225 23.894 −3.039 3220 HIS409 C −8.423 24.827 −2.0923221 HIS409 O −7.786 25.794 −2.516 3222 HIS409 CB −9.749 24.675 −4.2613223 HIS409 CG −10.004 23.779 −5.431 3224 HIS409 ND1 −11.239 23.595 −63225 HIS409 CD2 −9.152 23.013 −6.2 3226 HIS409 CE1 −11.089 22.715 −7.0373227 HIS409 NE2 −9.837 22.33 −7.181 3228 ARG410 N −8.27 24.332 −0.8083229 ARG410 CA −7.107 24.629 0.028 3230 ARG410 C −5.953 23.707 −0.4223231 ARG410 O −4.786 24.104 −0.504 3232 ARG410 CB −7.462 24.385 1.5 3233ARG410 CG −6.329 24.746 2.464 3234 ARG410 CD −6.767 24.581 3.916 3235ARG410 NE −5.679 24.889 4.856 3236 ARG410 CZ −4.671 24.047 5.193 3237ARG410 NH1 −4.554 22.815 4.655 3238 ARG410 NH2 −3.751 24.446 6.098 3239CYS411 N −6.287 22.387 −0.683 3240 CYS411 CA −5.272 21.47 −1.206 3241CYS411 C −4.915 21.959 −2.62 3242 CYS411 O −5.71 22.596 −3.317 3243CYS411 CB −5.753 20.019 −1.281 3244 CYS411 SG −6.127 19.345 0.368 3245ARG412 N −3.614 21.698 −3.013 3246 ARG412 CA −3.076 22.247 −4.266 3247ARG412 C −3.132 21.185 −5.377 3248 ARG412 O −3.331 21.488 −6.55 3249ARG412 CB −1.624 22.739 −4.093 3250 ARG412 CG −1.472 24.264 −3.936 3251ARG412 CD −1.97 24.831 −2.609 3252 ARG412 NE −3.426 24.817 −2.508 3253ARG412 CZ −4.289 25.563 −3.227 3254 ARG412 NH1 −3.925 26.701 −3.841 3255ARG412 NH2 −5.553 25.134 −3.334 3256 LYS413 N −2.775 19.912 −4.979 3257LYS413 CA −2.423 18.864 −5.94 3258 LYS413 C −3.566 17.87 −6.208 3259LYS413 O −3.417 16.89 −6.934 3260 LYS413 CB −1.2 18.077 −5.447 3261LYS413 CG 0.061 18.939 −5.336 3262 LYS413 CD 1.276 18.072 −5.005 3263LYS413 CE 2.534 18.909 −4.838 3264 LYS413 NZ 3.687 18.012 −4.683 3265ARG414 N −4.745 18.116 −5.519 3266 ARG414 CA −5.96 17.328 −5.776 3267ARG414 C −5.757 15.849 −5.368 3268 ARG414 O −6.423 14.935 −5.854 3269ARG414 CB −6.46 17.417 −7.226 3270 ARG414 CG −6.939 18.805 −7.644 3271ARG414 CD −8.386 19.082 −7.238 3272 ARG414 NE −8.988 20.13 −8.069 3273ARG414 CZ −9.199 20.038 −9.405 3274 ARG414 NH1 −9.55 21.142 −10.09 3275ARG414 NH2 −9.091 18.866 −10.056 3276 LYS415 N −4.876 15.685 −4.308 3277LYS415 CA −4.204 14.401 −4.077 3278 LYS415 C −5.175 13.385 −3.436 3279LYS415 O −5.764 13.646 −2.38 3280 LYS415 CB −3.002 14.629 −3.139 3281LYS415 CG −2.124 13.386 −2.95 3282 LYS415 CD −0.881 13.715 −2.115 3283LYS415 CE −0.054 12.499 −1.707 3284 LYS415 NZ 0.445 11.776 −2.873 3285PRO416 N −5.289 12.137 −4.035 3286 PRO416 CA −6.208 11.119 −3.493 3287PRO416 C −5.699 10.44 −2.2 3288 PRO416 O −5.502 9.229 −2.116 3289 PRO416CB −6.379 10.111 −4.639 3290 PRO416 CG −6.094 10.922 −5.889 3291 PRO416CD −4.993 11.863 −5.44 3292 LEU417 N −5.558 11.296 −1.119 3293 LEU417 CA−5.151 10.828 0.207 3294 LEU417 C −6.354 10.546 1.155 3295 LEU417 O−7.531 10.596 0.797 3296 LEU417 CB −4.029 11.711 0.793 3297 LEU417 CG−4.428 12.987 1.563 3298 LEU417 CD1 −3.186 13.579 2.241 3299 LEU417 CD2−5.07 14.051 0.682 3300 LEU418 N −5.985 10.162 2.433 3301 LEU418 CA−6.93 9.736 3.481 3302 LEU418 C −7.686 10.961 4.037 3303 LEU418 O −7.21412.1 4.002 3304 LEU418 CB −6.102 9.081 4.61 3305 LEU418 CG −6.88 8.3325.706 3306 LEU418 CD1 −7.557 7.083 5.159 3307 LEU418 CD2 −5.933 7.9376.844 3308 ALA419 N −8.902 10.672 4.638 3309 ALA419 CA −9.558 11.6295.528 3310 ALA419 C −9.125 11.34 6.98 3311 ALA419 O −8.25 12 7.538 3312ALA419 CB −11.072 11.637 5.361 3313 ILE420 N −9.751 10.262 7.572 3314ILE420 CA −9.394 9.73 8.889 3315 ILE420 C −9.594 8.204 8.777 3316 ILE420O −10.033 7.702 7.74 3317 ILE420 CB −10.195 10.35 10.062 3318 ILE420 CG1−11.716 10.074 10.052 3319 ILE420 CG2 −9.889 11.838 10.256 3320 ILE420CD1 −12.536 10.817 9.009 3321 GLY421 N −9.242 7.453 9.882 3322 GLY421 CA−9.545 6.029 9.923 3323 GLY421 C −10.876 5.785 10.639 3324 GLY421 O−11.293 6.56 11.506 3325 PHE422 N −11.516 4.611 10.28 3326 PHE422 CA−12.881 4.271 10.723 3327 PHE422 C −12.978 2.908 11.459 3328 PHE422 O−12.03 2.131 11.58 3329 PHE422 CB −13.905 4.426 9.586 3330 PHE422 CG−13.725 3.469 8.435 3331 PHE422 CD1 −14.505 2.311 8.335 3332 PHE422 CD2−12.772 3.732 7.443 3333 PHE422 CE1 −14.332 1.436 7.261 3334 PHE422 CE2−12.596 2.855 6.375 3335 PHE422 CZ −13.376 1.708 6.286 3336 TYR423 N−14.215 2.649 12.042 3337 TYR423 CA −14.36 1.647 13.112 3338 TYR423 C−15.679 0.909 12.861 3339 TYR423 O −16.769 1.43 13.102 3340 TYR423 CB−14.392 2.292 14.525 3341 TYR423 CG −13.436 3.454 14.663 3342 TYR423 CD1−13.908 4.752 14.408 3343 TYR423 CD2 −12.065 3.241 14.853 3344 TYR423CE1 −13.014 5.801 14.239 3345 TYR423 CE2 −11.166 4.292 14.683 3346TYR423 CZ −11.652 5.542 14.327 3347 TYR423 OH −10.774 6.499 13.905 3348LEU424 N −15.555 −0.326 12.252 3349 LEU424 CA −16.735 −1.139 11.976 3350LEU424 C −17.057 −2.062 13.172 3351 LEU424 O −16.209 −2.733 13.761 3352LEU424 CB −16.547 −2.012 10.728 3353 LEU424 CG −16.532 −1.242 9.395 3354LEU424 CD1 −16.122 −2.192 8.266 3355 LEU424 CD2 −17.888 −0.613 9.07 3356TYR425 N −18.407 −2.132 13.465 3357 TYR425 CA −18.935 −2.946 14.562 3358TYR425 C −20.066 −3.78 13.949 3359 TYR425 O −21.154 −3.292 13.65 3360TYR425 CB −19.461 −2.075 15.711 3361 TYR425 CG −18.348 −1.351 16.4363362 TYR425 CD1 −18.103 0.007 16.184 3363 TYR425 CD2 −17.52 −2.03717.337 3364 TYR425 CE1 −17.053 0.67 16.823 3365 TYR425 CE2 −16.465−1.376 17.97 3366 TYR425 CZ −16.239 −0.029 17.709 3367 TYR425 OH −15.1950.579 18.343 3368 ARG426 N −19.7 −5.072 13.62 3369 ARG426 CA −20.619−5.985 12.935 3370 ARG426 C −21.328 −6.886 13.962 3371 ARG426 O −20.791−7.22 15.016 3372 ARG426 CB −19.831 −6.833 11.928 3373 ARG426 CG −19.498−6.044 10.651 3374 ARG426 CD −18.185 −6.492 10.014 3375 ARG426 NE−18.271 −6.602 8.552 3376 ARG426 CZ −18.851 −7.644 7.904 3377 ARG426 NH1−18.874 −7.652 6.555 3378 ARG426 NH2 −19.409 −8.672 8.574 3379 MET427 N−22.589 −7.326 13.584 3380 MET427 CA −23.458 −8.081 14.497 3381 MET427 C−24.524 −8.836 13.668 3382 MET427 O −25.435 −8.232 13.105 3383 MET427 CB−24.21 −7.149 15.474 3384 MET427 CG −23.321 −6.538 16.555 3385 MET427 SD−24.334 −5.591 17.739 3386 MET427 CE −23.003 −4.993 18.809 3387 ASN428 N−24.345 −10.21 13.56 3388 ASN428 CA −25.495 −11.121 13.342 3389 ASN428 C−26.198 −10.914 11.983 3390 ASN428 O −27.267 −10.315 11.873 3391 ASN428CB −26.504 −11.046 14.496 3392 ASN428 CG −25.841 −11.268 15.835 3393ASN428 OD1 −25.592 −10.354 16.617 3394 ASN428 ND2 −25.496 −12.557 16.1163395 LYS429 N −25.502 −11.41 10.89 3396 LYS429 CA −25.762 −10.926 9.5293397 LYS429 C −25.484 −12.015 8.456 3398 LYS429 O −25.267 −13.184 8.7673399 LYS429 CB −24.914 −9.658 9.352 3400 LYS429 CG −25.731 −8.384 9.5633401 LYS429 CD −24.844 −7.196 9.938 3402 LYS429 CE −25.496 −5.884 9.553403 LYS429 NZ −25.222 −5.639 8.124 3404 GLU430 N −25.568 −11.583 7.1323405 GLU430 CA −25.522 −12.497 5.971 3406 GLU430 C −24.303 −12.271 5.0333407 GLU430 O −23.738 −11.184 4.938 3408 GLU430 CB −26.789 −12.36 5.123409 GLU430 CG −28.058 −12.739 5.875 3410 GLU430 CD −29.177 −12.7474.838 3411 GLU430 OE1 −29.853 −11.683 4.78 3412 GLU430 OE2 −29.261−13.809 4.158 3413 MET431 N −23.937 −13.375 4.265 3414 MET431 CA −22.64−13.472 3.578 3415 MET431 C −22.448 −12.481 2.41 3416 MET431 O −23.38−11.916 1.837 3417 MET431 CB −22.355 −14.908 3.081 3418 MET431 CG−21.332 −15.608 3.982 3419 MET431 SD −21.075 −17.319 3.462 3420 MET431CE −19.889 −17.834 4.72 3421 THR432 N −21.117 −12.353 2.026 3422 THR432CA −20.633 −11.428 1 3423 THR432 C −20.416 −12.185 −0.332 3424 THR432 O−21.316 −12.297 −1.171 3425 THR432 CB −19.345 −10.708 1.456 3426 THR432OG1 −18.337 −11.689 1.747 3427 THR432 CG2 −19.559 −9.846 2.692 3428TRP433 N −19.203 −12.821 −0.466 3429 TRP433 CA −18.684 −13.334 −1.7413430 TRP433 C −17.643 −14.447 −1.47 3431 TRP433 O −17.445 −14.869 −0.333432 TRP433 CB −18.203 −12.196 −2.66 3433 TRP433 CG −17.287 −11.209−2.005 3434 TRP433 CD1 −16.045 −11.47 −1.461 3435 TRP433 CD2 −17.527−9.805 −1.836 3436 TRP433 NE1 −15.553 −10.308 −0.933 3437 TRP433 CE2−16.424 −9.273 −1.171 3438 TRP433 CE3 −18.578 −8.933 −2.188 3439 TRP433CZ2 −16.306 −7.916 −0.848 3440 TRP433 CZ3 −18.473 −7.572 −1.876 3441TRP433 CH2 −17.352 −7.072 −1.22 3442 SER434 N −17.074 −14.988 −2.6073443 SER434 CA −16.302 −16.245 −2.675 3444 SER434 C −14.816 −16.052−2.31 3445 SER434 O −14.388 −15.017 −1.798 3446 SER434 CB −16.474−16.814 −4.092 3447 SER434 OG −16.026 −15.858 −5.058 3448 SER435 N−13.978 −17.125 −2.611 3449 SER435 CA −12.701 −17.345 −1.906 3450 SER435C −11.638 −16.241 −1.942 3451 SER435 O −10.671 −16.273 −1.174 3452SER435 CB −12.027 −18.653 −2.353 3453 SER435 OG −11.566 −18.579 −3.7063454 LEU436 N −11.87 −15.187 −2.797 3455 LEU436 CA −11.111 −13.952 −2.653456 LEU436 C −11.359 −13.328 −1.26 3457 LEU436 O −10.531 −12.606 −0.7053458 LEU436 CB −11.517 −12.903 −3.684 3459 LEU436 CG −11.315 −13.279−5.154 3460 LEU436 CD1 −11.864 −12.154 −6.032 3461 LEU436 CD2 −9.846−13.513 −5.489 3462 GLY437 N −12.62 −13.556 −0.747 3463 GLY437 CA−13.039 −13.084 0.547 3464 GLY437 C −12.719 −14.037 1.689 3465 GLY437 O−13.079 −13.791 2.842 3466 SER438 N −11.88 −15.091 1.39 3467 SER438 CA−11.446 −16.058 2.416 3468 SER438 C −10.602 −15.312 3.458 3469 SER438 O−10.428 −15.716 4.604 3470 SER438 CB −10.628 −17.205 1.821 3471 SER438OG −9.526 −16.709 1.058 3472 ARG439 N −10.038 −14.168 2.939 3473 ARG439CA −9.286 −13.194 3.686 3474 ARG439 C −10.124 −11.915 3.874 3475 ARG439O −9.563 −10.825 3.969 3476 ARG439 CB −7.99 −12.932 2.92 3477 ARG439 CG−6.867 −12.299 3.739 3478 ARG439 CD −5.585 −12.165 2.925 3479 ARG439 NE−5.069 −13.475 2.519 3480 ARG439 CZ −5.407 −14.133 1.391 3481 ARG439 NH1−5.1 −15.423 1.213 3482 ARG439 NH2 −6.106 −13.565 0.399 3483 GLN440 N−11.463 −12.096 4.166 3484 GLN440 CA −12.465 −11.035 4.403 3485 GLN440 C−12.178 −10.022 5.552 3486 GLN440 O −11.043 −9.693 5.896 3487 GLN440 CB−12.943 −10.352 3.108 3488 GLN440 CG −11.864 −9.689 2.259 3489 GLN440 CD−12.414 −9.178 0.948 3490 GLN440 OE1 −13.474 −9.548 0.455 3491 GLN440NE2 −11.597 −8.285 0.317 3492 PRO441 N −13.239 −9.428 6.202 3493 PRO441CA −13.036 −8.136 6.862 3494 PRO441 C −12.416 −8.117 8.287 3495 PRO441 O−13.114 −7.991 9.295 3496 PRO441 CB −14.447 −7.52 6.857 3497 PRO441 CG−15.365 −8.731 6.917 3498 PRO441 CD −14.652 −9.738 6.038 3499 PHE442 N−11.029 −8.153 8.366 3500 PHE442 CA −10.332 −7.805 9.62 3501 PHE442 C−9.083 −6.896 9.496 3502 PHE442 O −9.206 −5.679 9.672 3503 PHE442 CB−10.116 −8.98 10.59 3504 PHE442 CG −9.39 −8.563 11.855 3505 PHE442 CD1−9.915 −7.563 12.689 3506 PHE442 CD2 −8.13 −9.099 12.155 3507 PHE442 CE1−9.175 −7.076 13.767 3508 PHE442 CE2 −7.399 −8.621 13.245 3509 PHE442 CZ−7.914 −7.601 14.042 3510 PHE443 N −7.864 −7.47 9.167 3511 PHE443 CA−6.592 −6.816 9.559 3512 PHE443 C −6.519 −5.329 9.168 3513 PHE443 O−6.065 −4.502 9.956 3514 PHE443 CB −5.35 −7.501 8.958 3515 PHE443 CG−4.915 −8.78 9.632 3516 PHE443 CD1 −4.499 −8.779 10.97 3517 PHE443 CD2−4.845 −9.977 8.906 3518 PHE443 CE1 −4.053 −9.956 11.575 3519 PHE443 CE2−4.392 −11.152 9.509 3520 PHE443 CZ −4.002 −11.141 10.845 3521 SER444 N−6.944 −5.024 7.889 3522 SER444 CA −7.273 −3.663 7.462 3523 SER444 C−6.103 −2.826 6.927 3524 SER444 O −4.948 −2.922 7.329 3525 SER444 CB−8.006 −2.808 8.523 3526 SER444 OG −9.277 −3.34 8.873 3527 LEU445 N−6.519 −1.872 6.004 3528 LEU445 CA −5.909 −0.544 5.962 3529 LEU445 C−7.064 0.398 6.361 3530 LEU445 O −8.241 0.056 6.242 3531 LEU445 CB−5.414 −0.178 4.561 3532 LEU445 CG −4.232 −1.026 4.061 3533 LEU445 CD1−3.967 −0.717 2.587 3534 LEU445 CD2 −2.959 −0.783 4.872 3535 GLU446 N−6.68 1.619 6.875 3536 GLU446 CA −7.618 2.736 7.041 3537 GLU446 C −8.7122.552 8.119 3538 GLU446 O −9.611 3.382 8.283 3539 GLU446 CB −8.281 3.1875.724 3540 GLU446 CG −7.293 3.445 4.588 3541 GLU446 CD −7.919 4.0793.345 3542 GLU446 OE1 −7.238 3.967 2.289 3543 GLU446 OE2 −9.047 4.6373.496 3544 ALA447 N −8.607 1.443 8.935 3545 ALA447 CA −9.769 1.048 9.7163546 ALA447 C −9.445 0.063 10.841 3547 ALA447 O −8.421 −0.612 10.8643548 ALA447 CB −10.827 0.409 8.815 3549 CYS448 N −10.472 −0.034 11.7663550 CYS448 CA −10.613 −1.163 12.675 3551 CYS448 C −11.992 −1.8 12.413552 CYS448 O −12.953 −1.175 11.954 3553 CYS448 CB −10.567 −0.751 14.153554 CYS448 SG −8.969 −0.041 14.652 3555 GLN449 N −12.101 −3.113 12.8283556 GLN449 CA −13.353 −3.852 12.697 3557 GLN449 C −13.478 −4.789 13.9013558 GLN449 O −12.505 −5.372 14.383 3559 GLN449 CB −13.392 −4.607 11.363560 GLN449 CG −14.686 −5.391 11.114 3561 GLN449 CD −14.77 −6.726 11.8273562 GLN449 OE1 −15.787 −7.134 12.383 3563 GLN449 NE2 −13.64 −7.48611.733 3564 GLY450 N −14.782 −4.982 14.336 3565 GLY450 CA −15.039 −5.92215.4 3566 GLY450 C −16.488 −6.393 15.59 3567 GLY450 O −17.472 −5.87815.069 3568 ILE451 N −16.545 −7.431 16.511 3569 ILE451 CA −17.782 −7.96717.084 3570 ILE451 C −17.402 −8.498 18.495 3571 ILE451 O −16.283 −8.96918.747 3572 ILE451 CB −18.431 −9.052 16.18 3573 ILE451 CG1 −19.753−9.581 16.776 3574 ILE451 CG2 −17.474 −10.212 15.914 3575 ILE451 CD1−20.564 −10.47 15.844 3576 LEU452 N −18.429 −8.406 19.42 3577 LEU452 CA−18.457 −9.171 20.67 3578 LEU452 C −19.38 −10.365 20.366 3579 LEU452 O−20.564 −10.223 20.049 3580 LEU452 CB −19.03 −8.361 21.838 3581 LEU452CG −17.969 −7.553 22.614 3582 LEU452 CD1 −17.285 −6.489 21.757 3583LEU452 CD2 −18.613 −6.897 23.838 3584 ALA453 N −18.72 −11.575 20.3343585 ALA453 CA −19.368 −12.823 19.955 3586 ALA453 C −19.845 −13.57321.207 3587 ALA453 O −19.208 −13.543 22.263 3588 ALA453 CB −18.397−13.666 19.146 3589 LEU454 N −20.992 −14.335 21.039 3590 LEU454 CA−21.678 −14.884 22.218 3591 LEU454 C −22.532 −16.117 21.838 3592 LEU454O −23.352 −16.077 20.924 3593 LEU454 CB −22.578 −13.791 22.84 3594LEU454 CG −22.179 −13.341 24.254 3595 LEU454 CD1 −23.059 −12.174 24.7023596 LEU454 CD2 −22.264 −14.459 25.287 3597 LEU455 N −22.303 −17.23222.626 3598 LEU455 CA −23.037 −18.504 22.562 3599 LEU455 C −22.923−19.278 21.221 3600 LEU455 O −21.809 −19.515 20.749 3601 LEU455 CB−24.366 −18.564 23.336 3602 LEU455 CG −25.471 −17.604 22.881 3603 LEU455CD1 −26.844 −18.245 23.061 3604 LEU455 CD2 −25.453 −16.307 23.685 3605ASP456 N −24.08 −19.73 20.622 3606 ASP456 CA −24.079 −20.807 19.612 3607ASP456 C −25.142 −20.552 18.52 3608 ASP456 O −25.977 −21.383 18.16 3609ASP456 CB −24.277 −22.157 20.297 3610 ASP456 CG −25.561 −22.276 21.1173611 ASP456 OD1 −25.678 −23.357 21.757 3612 ASP456 OD2 −26.351 −21.28221.082 3613 LEU457 N −25.039 −19.315 17.918 3614 LEU457 CA −26.163−18.744 17.205 3615 LEU457 C −26.23 −19.102 15.711 3616 LEU457 O −25.275−18.996 14.942 3617 LEU457 CB −26.152 −17.21 17.303 3618 LEU457 CG−26.172 −16.655 18.737 3619 LEU457 CD1 −26.215 −15.13 18.687 3620 LEU457CD2 −27.342 −17.195 19.553 3621 ASN458 N −27.517 −19.404 15.283 3622ASN458 CA −27.869 −19.553 13.868 3623 ASN458 C −28.119 −18.161 13.2713624 ASN458 O −28.082 −17.963 12.057 3625 ASN458 CB −29.094 −20.43813.704 3626 ASN458 CG −29.345 −20.88 12.281 3627 ASN458 OD1 −30.369−20.599 11.654 3628 ASN458 ND2 −28.395 −21.678 11.711 3629 ALA459 N−28.339 −17.149 14.183 3630 ALA459 CA −28.623 −15.775 13.76 3631 ALA459C −27.383 −15.129 13.104 3632 ALA459 O −27.454 −14.087 12.457 3633ALA459 CB −29.048 −14.929 14.952 3634 SER460 N −26.191 −15.771 13.3773635 SER460 CA −24.923 −15.423 12.737 3636 SER460 C −24.465 −16.52311.762 3637 SER460 O −23.307 −16.571 11.336 3638 SER460 CB −23.826−15.211 13.796 3639 SER460 OG −24.097 −14.046 14.573 3640 GLY461 N−25.421 −17.425 11.328 3641 GLY461 CA −24.972 −18.751 10.942 3642 GLY461C −26.015 −19.652 10.301 3643 GLY461 O −26.07 −20.852 10.568 3644 THR462N −26.747 −19.036 9.319 3645 THR462 CA −27.6 −19.729 8.332 3646 THR462 C−27.351 −18.909 7.069 3647 THR462 O −27.307 −17.676 7.15 3648 THR462 CB−29.076 −19.719 8.781 3649 THR462 OG1 −29.416 −21.025 9.273 3650 THR462CG2 −30.111 −19.384 7.717 3651 MET463 N −27.18 −19.512 5.856 3652 MET463CA −27.327 −20.912 5.478 3653 MET463 C −26.033 −21.723 5.745 3654 MET463O −25.904 −22.367 6.789 3655 MET463 CB −27.787 −21.064 4.019 3656 MET463CG −29.23 −20.624 3.748 3657 MET463 SD −29.329 −19.017 2.886 3658 MET463CE −29.982 −17.973 4.209 3659 SER464 N −25.026 −21.675 4.795 3660 SER464CA −23.925 −22.653 4.837 3661 SER464 C −22.64 −22.062 4.223 3662 SER464O −22.496 −20.845 4.081 3663 SER464 CB −24.372 −23.959 4.164 3664 SER464OG −24.026 −24.012 2.783 3665 ILE465 N −21.628 −22.979 3.953 3666 ILE465CA −20.496 −22.62 3.087 3667 ILE465 C −20.695 −23.153 1.652 3668 ILE465O −19.948 −22.821 0.73 3669 ILE465 CB −19.145 −23.079 3.699 3670 ILE465CG1 −17.945 −22.428 2.976 3671 ILE465 CG2 −19 −24.602 3.768 3672 ILE465CD1 −16.65 −22.488 3.777 3673 GLN466 N −21.729 −24.05 1.464 3674 GLN466CA −21.953 −24.678 0.166 3675 GLN466 C −22.583 −23.712 −0.858 3676GLN466 O −22.796 −24.041 −2.023 3677 GLN466 CB −22.784 −25.961 0.2893678 GLN466 CG −22.141 −26.997 1.216 3679 GLN466 CD −22.575 −26.8322.658 3680 GLN466 OE1 −21.911 −26.247 3.509 3681 GLN466 NE2 −23.792−27.386 2.948 3682 GLU467 N −22.787 −22.432 −0.379 3683 GLU467 CA−23.107 −21.313 −1.25 3684 GLU467 C −21.82 −20.77 −1.92 3685 GLU467 O−21.881 −19.958 −2.844 3686 GLU467 CB −23.776 −20.18 −0.456 3687 GLU467CG −25.224 −20.493 −0.071 3688 GLU467 CD −25.479 −21.591 0.957 3689GLU467 OE1 −26.685 −21.909 1.125 3690 GLU467 OE2 −24.462 −22.076 1.5493691 PHE468 N −20.625 −21.177 −1.354 3692 PHE468 CA −19.297 −20.857−1.887 3693 PHE468 C −18.973 −19.378 −1.635 3694 PHE468 O −18.508−18.641 −2.502 3695 PHE468 CB −19.081 −21.242 −3.361 3696 PHE468 CG−19.031 −22.735 −3.584 3697 PHE468 CD1 −20.188 −23.444 −3.925 3698PHE468 CD2 −17.824 −23.433 −3.437 3699 PHE468 CE1 −20.141 −24.826 −4.1183700 PHE468 CE2 −17.777 −24.814 −3.637 3701 PHE468 CZ −18.935 −25.51−3.977 3702 ARG469 N −19.176 −18.978 −0.326 3703 ARG469 CA −18.955−17.606 0.138 3704 ARG469 C −18.233 −17.68 1.516 3705 ARG469 O −18.179−18.736 2.152 3706 ARG469 CB −20.289 −16.841 0.146 3707 ARG469 CG−20.949 −16.705 −1.238 3708 ARG469 CD −22.418 −16.286 −1.154 3709 ARG469NE −22.576 −14.835 −1.038 3710 ARG469 CZ −23.571 −14.191 −0.388 3711ARG469 NH1 −23.577 −12.85 −0.416 3712 ARG469 NH2 −24.531 −14.828 0.3093713 ASP470 N −17.645 −16.493 1.966 3714 ASP470 CA −16.41 −16.571 2.7973715 ASP470 C −16.38 −15.736 4.16 3716 ASP470 O −17.401 −15.304 4.6883717 ASP470 CB −15.244 −16.262 1.861 3718 ASP470 CG −14.765 −17.5491.208 3719 ASP470 OD1 −13.791 −18.098 1.798 3720 ASP470 OD2 −15.341−17.873 0.133 3721 LEU471 N −15.118 −15.643 4.759 3722 LEU471 CA −14.762−15.476 6.201 3723 LEU471 C −15.071 −14.186 7.029 3724 LEU471 O −15.89−13.351 6.66 3725 LEU471 CB −15.013 −16.798 6.953 3726 LEU471 CG −15.999−16.857 8.125 3727 LEU471 CD1 −17.41 −16.426 7.758 3728 LEU471 CD2−16.021 −18.296 8.651 3729 TRP472 N −14.336 −14.066 8.234 3730 TRP472 CA−14.807 −13.29 9.424 3731 TRP472 C −13.809 −13.453 10.645 3732 TRP472 O−13.539 −14.592 11.04 3733 TRP472 CB −15.274 −11.864 9.136 3734 TRP472CG −16.065 −11.203 10.232 3735 TRP472 CD1 −15.809 −9.925 10.672 3736TRP472 CD2 −17.245 −11.631 10.929 3737 TRP472 NE1 −16.69 −9.598 11.663738 TRP472 CE2 −17.645 −10.577 11.759 3739 TRP472 CE3 −18.065 −12.77910.891 3740 TRP472 CZ2 −18.831 −10.599 12.5 3741 TRP472 CZ3 −19.274−12.8 11.599 3742 TRP472 CH2 −19.649 −11.725 12.395 3743 LYS473 N−13.317 −12.328 11.277 3744 LYS473 CA −12.528 −12.244 12.559 3745 LYS473C −12.555 −10.73 12.919 3746 LYS473 O −13.222 −9.962 12.208 3747 LYS473CB −11.089 −12.779 12.405 3748 LYS473 CG −10.658 −13.618 13.62 3749LYS473 CD −9.176 −13.615 14.033 3750 LYS473 CE −8.406 −12.325 13.8053751 LYS473 NZ −7.253 −12.238 14.705 3752 GLN474 N −11.902 −10.12513.948 3753 GLN474 CA −11.485 −10.565 15.29 3754 GLN474 C −12.758−10.505 16.136 3755 GLN474 O −13.294 −9.445 16.479 3756 GLN474 CB−10.453 −9.581 15.879 3757 GLN474 CG −10.067 −9.819 17.345 3758 GLN474CD −8.796 −10.613 17.546 3759 GLN474 OE1 −8.217 −11.236 16.662 3760GLN474 NE2 −8.339 −10.619 18.834 3761 LEU475 N −13.276 −11.753 16.4253762 LEU475 CA −14.466 −11.861 17.241 3763 LEU475 C −14.037 −12.15618.68 3764 LEU475 O −13.301 −13.098 18.97 3765 LEU475 CB −15.503 −12.84416.703 3766 LEU475 CG −15.106 −14.314 16.529 3767 LEU475 CD1 −16.382−15.157 16.439 3768 LEU475 CD2 −14.241 −14.554 15.294 3769 LYS476 N−14.52 −11.229 19.594 3770 LYS476 CA −14.296 −11.408 21.023 3771 LYS476C −15.403 −12.373 21.456 3772 LYS476 O −16.557 −11.986 21.643 3773LYS476 CB −14.448 −10.084 21.787 3774 LYS476 CG −13.182 −9.226 21.8713775 LYS476 CD −13.144 −8.045 20.896 3776 LYS476 CE −12.76 −8.445 19.4833777 LYS476 NZ −13.864 −8.18 18.544 3778 LEU477 N −15.034 −13.706 21.4753779 LEU477 CA −15.975 −14.735 21.911 3780 LEU477 C −16.033 −14.62523.426 3781 LEU477 O −15.074 −14.88 24.151 3782 LEU477 CB −15.534−16.129 21.469 3783 LEU477 CG −16.481 −17.3 21.82 3784 LEU477 CD1 −16.3−17.827 23.24 3785 LEU477 CD2 −17.957 −17.001 21.566 3786 SER478 N−17.236 −14.142 23.884 3787 SER478 CA −17.475 −13.916 25.289 3788 SER478C −17.836 −15.275 25.896 3789 SER478 O −18.871 −15.882 25.619 3790SER478 CB −18.596 −12.9 25.497 3791 SER478 OG −18.322 −12.077 26.6463792 GLN479 N −16.886 −15.779 26.773 3793 GLN479 CA −17.03 −17.108 27.393794 GLN479 C −18.077 −16.997 28.527 3795 GLN479 O −17.791 −17.04929.722 3796 GLN479 CB −15.688 −17.598 27.959 3797 GLN479 CG −14.69−18.011 26.879 3798 GLN479 CD −14.908 −19.453 26.483 3799 GLN479 OE1−15.734 −19.8 25.647 3800 GLN479 NE2 −14.156 −20.36 27.173 3801 LYS480 N−19.356 −16.773 28.067 3802 LYS480 CA −20.517 −16.487 28.906 3803 LYS480C −21.77 −16.665 28.014 3804 LYS480 O −21.677 −17.136 26.877 3805 LYS480CB −20.356 −15.172 29.687 3806 LYS480 CG −19.899 −13.945 28.877 3807LYS480 CD −18.895 −13.069 29.654 3808 LYS480 CE −17.427 −13.47 29.4783809 LYS480 NZ −16.768 −12.661 28.431 3810 VAL481 N −22.997 −16.42728.604 3811 VAL481 CA −24.229 −17.002 28.043 3812 VAL481 C −25.429−16.015 28.089 3813 VAL481 O −25.351 −14.874 28.538 3814 VAL481 CB−24.567 −18.365 28.717 3815 VAL481 CG1 −23.517 −19.439 28.415 3816VAL481 CG2 −24.767 −18.237 30.229 3817 PHE482 N −26.597 −16.56 27.5713818 PHE482 CA −27.802 −15.775 27.256 3819 PHE482 C −28.301 −14.98928.477 3820 PHE482 O −28.668 −15.532 29.517 3821 PHE482 CB −28.909−16.748 26.797 3822 PHE482 CG −30.248 −16.149 26.442 3823 PHE482 CD1−30.36 −15.064 25.568 3824 PHE482 CD2 −31.424 −16.738 26.933 3825 PHE482CE1 −31.614 −14.568 25.206 3826 PHE482 CE2 −32.678 −16.249 26.563 3827PHE482 CZ −32.773 −15.165 25.696 3828 HIS483 N −28.23 −13.613 28.3113829 HIS483 CA −28.735 −12.668 29.304 3830 HIS483 C −27.97 −12.72230.637 3831 HIS483 O −28.45 −12.283 31.683 3832 HIS483 CB −30.254 −12.7429.528 3833 HIS483 CG −31.073 −12.26 28.373 3834 HIS483 ND1 −32.394−12.607 28.232 3835 HIS483 CD2 −30.826 −11.421 27.306 3836 HIS483 CE1−32.878 −11.963 27.13 3837 HIS483 NE2 −31.948 −11.251 26.535 3838 LYS484N −26.657 −13.133 30.533 3839 LYS484 CA −25.685 −12.862 31.58 3840LYS484 C −24.653 −11.904 30.972 3841 LYS484 O −24.407 −11.865 29.7673842 LYS484 CB −25.05 −14.143 32.123 3843 LYS484 CG −26.115 −15.06932.724 3844 LYS484 CD −25.513 −16.271 33.448 3845 LYS484 CE −26.619−17.214 33.907 3846 LYS484 NZ −26.018 −18.377 34.601 3847 GLN485 N−24.023 −11.091 31.9 3848 GLN485 CA −23.392 −9.862 31.435 3849 GLN485 C−22.075 −10.171 30.7 3850 GLN485 O −21.35 −11.127 30.976 3851 GLN485 CB−23.103 −8.888 32.593 3852 GLN485 CG −24.353 −8.174 33.122 3853 GLN485CD −25.272 −9.063 33.935 3854 GLN485 OE1 −25.095 −10.268 34.093 3855GLN485 NE2 −26.344 −8.412 34.475 3856 ASP486 N −21.755 −9.213 29.7443857 ASP486 CA −20.474 −9.264 29.055 3858 ASP486 C −19.431 −8.63 29.9853859 ASP486 O −19.696 −7.757 30.808 3860 ASP486 CB −20.494 −8.536 27.7183861 ASP486 CG −20.864 −9.533 26.626 3862 ASP486 OD1 −21.919 −9.2725.974 3863 ASP486 OD2 −20.035 −10.491 26.497 3864 ARG487 N −18.167−9.147 29.798 3865 ARG487 CA −17.035 −8.814 30.649 3866 ARG487 C −15.801−9.489 30.029 3867 ARG487 O −15.896 −10.495 29.316 3868 ARG487 CB−17.223 −9.265 32.114 3869 ARG487 CG −17.552 −10.755 32.235 3870 ARG487CD −17.844 −11.202 33.664 3871 ARG487 NE −18.195 −12.636 33.704 3872ARG487 CZ −17.357 −13.643 33.349 3873 ARG487 NH1 −17.852 −14.883 33.1533874 ARG487 NH2 −16.047 −13.438 33.161 3875 GLY488 N −14.596 −8.92630.413 3876 GLY488 CA −13.358 −9.294 29.739 3877 GLY488 C −12.713−10.564 30.276 3878 GLY488 O −11.803 −11.146 29.685 3879 SER489 N−13.197 −10.993 31.488 3880 SER489 CA −12.728 −12.203 32.159 3881 SER489C −13.311 −13.449 31.456 3882 SER489 O −14.069 −14.252 32.002 3883SER489 CB −13.068 −12.139 33.651 3884 SER489 OG −14.433 −11.726 33.8653885 GLY490 N −12.87 −13.583 30.151 3886 GLY490 CA −13.229 −14.693 29.33887 GLY490 C −13.465 −14.203 27.871 3888 GLY490 O −14.604 −13.94227.472 3889 TYR491 N −12.286 −14.06 27.158 3890 TYR491 CA −12.146−13.768 25.722 3891 TYR491 C −10.925 −14.598 25.239 3892 TYR491 O−10.081 −15.004 26.04 3893 TYR491 CB −11.825 −12.287 25.44 3894 TYR491CG −12.903 −11.243 25.626 3895 TYR491 CD1 −12.514 −9.911 25.86 3896TYR491 CD2 −14.265 −11.515 25.474 3897 TYR491 CE1 −13.461 −8.888 25.9633898 TYR491 CE2 −15.218 −10.499 25.599 3899 TYR491 CZ −14.811 −9.19425.84 3900 TYR491 OH −15.774 −8.234 25.938 3901 LEU492 N −10.827 −14.78623.871 3902 LEU492 CA −9.715 −15.511 23.231 3903 LEU492 C −9.789 −15.32421.697 3904 LEU492 O −10.873 −15.114 21.145 3905 LEU492 CB −9.668−17.007 23.602 3906 LEU492 CG −10.781 −17.923 23.041 3907 LEU492 CD1−10.571 −19.357 23.543 3908 LEU492 CD2 −12.197 −17.488 23.413 3909ASN493 N −8.603 −15.444 20.997 3910 ASN493 CA −8.429 −15.381 19.514 3911ASN493 C −6.895 −15.31 19.247 3912 ASN493 O −6.15 −14.752 20.061 3913ASN493 CB −9.05 −14.117 18.881 3914 ASN493 CG −10.391 −14.229 18.1653915 ASN493 OD1 −10.682 −13.55 17.18 3916 ASN493 ND2 −11.348 −15.02918.73 3917 TRP494 N −6.359 −15.716 18.044 3918 TRP494 CA −6.597 −17.00817.376 3919 TRP494 C −6.2 −16.972 15.869 3920 TRP494 O −7.085 −17.04315.008 3921 TRP494 CB −5.864 −18.162 18.077 3922 TRP494 CG −6.493−18.602 19.362 3923 TRP494 CD1 −7.744 −19.168 19.512 3924 TRP494 CD2−5.893 −18.577 20.663 3925 TRP494 NE1 −7.895 −19.543 20.819 3926 TRP494CE2 −6.784 −19.19 21.546 3927 TRP494 CE3 −4.659 −18.117 21.169 3928TRP494 CZ2 −6.494 −19.381 22.902 3929 TRP494 CZ3 −4.361 −18.287 22.5263930 TRP494 CH2 −5.266 −18.913 23.378 3931 GLU495 N −4.845 −16.90315.565 3932 GLU495 CA −4.323 −16.498 14.235 3933 GLU495 C −4.049 −17.66213.21 3934 GLU495 O −3.999 −18.834 13.572 3935 GLU495 CB −3.105 −15.57314.392 3936 GLU495 CG −3.338 −14.37 15.316 3937 GLU495 CD −4.475 −13.4714.863 3938 GLU495 OE1 −5.652 −13.786 15.24 3939 GLU495 OE2 −4.185−12.479 14.142 3940 GLN496 N −3.91 −17.286 11.867 3941 GLN496 CA −4.128−18.198 10.691 3942 GLN496 C −5.056 −17.459 9.649 3943 GLN496 O −4.914−16.249 9.455 3944 GLN496 CB −2.773 −18.562 10.06 3945 GLN496 CG −2.739−19.971 9.465 3946 GLN496 CD −3.606 −20.099 8.236 3947 GLN496 OE1 −4.785−20.438 8.302 3948 GLN496 NE2 −3.013 −19.72 7.067 3949 LEU497 N −6.036−18.212 9 3950 LEU497 CA −7.082 −17.633 8.107 3951 LEU497 C −8.492−18.334 8.207 3952 LEU497 O −8.592 −19.564 8.188 3953 LEU497 CB −6.683−17.756 6.624 3954 LEU497 CG −5.572 −16.816 6.133 3955 LEU497 CD1 −5.241−17.167 4.678 3956 LEU497 CD2 −5.983 −15.347 6.229 3957 HIS498 N −9.613−17.501 8.234 3958 HIS498 CA −11.051 −17.916 8.02 3959 HIS498 C −11.841−18.503 9.278 3960 HIS498 O −12.191 −19.685 9.35 3961 HIS498 CB −11.123−18.798 6.747 3962 HIS498 CG −12.453 −19.296 6.297 3963 HIS498 ND1−13.171 −20.239 6.994 3964 HIS498 CD2 −13.177 −19.116 5.137 3965 HIS498CE1 −14.275 −20.556 6.265 3966 HIS498 NE2 −14.325 −19.868 5.15 3967ALA499 N −12.13 −17.598 10.312 3968 ALA499 CA −12.588 −18.068 11.6493969 ALA499 C −14.09 −18.292 11.984 3970 ALA499 O −15.033 −17.654 11.5173971 ALA499 CB −12.103 −17.133 12.772 3972 ALA500 N −14.242 −19.21513.024 3973 ALA500 CA −15.505 −19.589 13.67 3974 ALA500 C −15.146−20.461 14.896 3975 ALA500 O −14.092 −20.275 15.507 3976 ALA500 CB−16.39 −20.34 12.698 3977 MET501 N −16.081 −21.397 15.283 3978 MET501 CA−15.902 −22.298 16.434 3979 MET501 C −15.979 −21.489 17.736 3980 MET501O −15.297 −21.742 18.725 3981 MET501 CB −14.634 −23.153 16.353 3982MET501 CG −14.778 −24.434 17.177 3983 MET501 SD −13.151 −25.163 17.5433984 MET501 CE −12.736 −24.159 18.999 3985 ARG502 N −16.942 −20.50517.697 3986 ARG502 CA −17.038 −19.447 18.694 3987 ARG502 C −18.557−19.172 18.805 3988 ARG502 O −19.316 −20.076 19.158 3989 ARG502 CB−16.146 −18.28 18.257 3990 ARG502 CG −14.626 −18.523 18.409 3991 ARG502CD −13.871 −17.382 17.745 3992 ARG502 NE −12.409 −17.446 17.791 3993ARG502 CZ −11.63 −18.186 16.968 3994 ARG502 NH1 −10.307 −17.933 16.923995 ARG502 NH2 −12.109 −19.143 16.165 3996 GLU503 N −19.034 −17.96418.301 3997 GLU503 CA −20.488 −17.651 18.368 3998 GLU503 C −21.257−18.617 17.459 3999 GLU503 O −22.454 −18.869 17.584 4000 GLU503 CB−20.749 −16.219 17.876 4001 GLU503 CG −22.228 −15.886 17.704 4002 GLU503CD −22.439 −14.387 17.63 4003 GLU503 OE1 −22.748 −13.938 16.486 4004GLU503 OE2 −22.316 −13.748 18.729 4005 ALA504 N −20.506 −19.008 16.3724006 ALA504 CA −20.935 −20.061 15.492 4007 ALA504 C −19.729 −20.98615.274 4008 ALA504 O −18.558 −20.607 15.372 4009 ALA504 CB −21.442−19.471 14.191 4010 GLY505 N −20.091 −22.262 14.917 4011 GLY505 CA−19.139 −23.332 14.703 4012 GLY505 C −19.814 −24.4 13.856 4013 GLY505 O−20.725 −24.12 13.072 4014 ARG506 N −19.264 −25.662 14.004 4015 ARG506CA −19.955 −26.86 13.504 4016 ARG506 C −20.693 −27.446 14.714 4017ARG506 O −20.065 −27.881 15.68 4018 ARG506 CB −18.946 −27.862 12.9384019 ARG506 CG −19.611 −29.153 12.447 4020 ARG506 CD −18.652 −30.02711.642 4021 ARG506 NE −18.39 −29.446 10.322 4022 ARG506 CZ −17.684−30.031 9.334 4023 ARG506 NH1 −17.591 −29.385 8.157 4024 ARG506 NH2−17.083 −31.226 9.486 4025 HIS507 N −22.069 −27.325 14.683 4026 HIS507CA −22.898 −27.667 15.848 4027 HIS507 C −24.408 −27.458 15.594 4028HIS507 O −24.865 −26.798 14.664 4029 HIS507 CB −22.481 −26.938 17.1574030 HIS507 CG −22.311 −25.457 17.051 4031 HIS507 ND1 −21.963 −24.67418.126 4032 HIS507 CD2 −22.406 −24.57 16.004 4033 HIS507 CE1 −21.806−23.393 17.674 4034 HIS507 NE2 −22.047 −23.299 16.384 4035 ARG508 N−25.233 −28.045 16.547 4036 ARG508 CA −26.681 −27.836 16.521 4037 ARG508C −26.953 −26.425 17.074 4038 ARG508 O −27.094 −26.198 18.273 4039ARG508 CB −27.41 −28.873 17.387 4040 ARG508 CG −27.241 −30.306 16.8814041 ARG508 CD −28.092 −31.173 17.698 4042 ARG508 NE −27.858 −32.67117.307 4043 ARG508 CZ −26.814 −33.431 17.719 4044 ARG508 NH1 −26.724−34.713 17.302 4045 ARG508 NH2 −25.855 −32.95 18.538 4046 LYS509 N−26.933 −25.445 16.096 4047 LYS509 CA −26.969 −24.029 16.467 4048 LYS509C −28.345 −23.634 17.061 4049 LYS509 O −29.398 −24.176 16.727 4050LYS509 CB −26.716 −23.15 15.232 4051 LYS509 CG −25.253 −23.169 14.7854052 LYS509 CD −25.024 −22.297 13.551 4053 LYS509 CE −23.562 −21.95713.328 4054 LYS509 NZ −22.88 −22.921 12.47 4055 SER510 N −28.295 −22.57317.952 4056 SER510 CA −29.498 −22.004 18.563 4057 SER510 C −30.006−20.796 17.746 4058 SER510 O −29.321 −20.221 16.898 4059 SER510 CB−29.268 −21.629 20.032 4060 SER510 OG −28.392 −20.517 20.163 4061 TRP511N −31.309 −20.407 18.039 4062 TRP511 CA −31.959 −19.289 17.34 4063TRP511 C −32.482 −18.271 18.358 4064 TRP511 O −32.101 −17.099 18.3664065 TRP511 CB −33.115 −19.757 16.431 4066 TRP511 CG −32.941 −19.31715.011 4067 TRP511 CD1 −32.722 −20.154 13.938 4068 TRP511 CD2 −32.94−17.976 14.5 4069 TRP511 NE1 −32.51 −19.383 12.827 4070 TRP511 CE2−32.641 −18.051 13.138 4071 TRP511 CE3 −33.131 −16.699 15.07 4072 TRP511CZ2 −32.5 −16.919 12.326 4073 TRP511 CZ3 −33.004 −15.557 14.271 4074TRP511 CH2 −32.69 −15.668 12.919 4075 SER512 N −33.435 −18.746 19.2444076 SER512 CA −34.294 −17.813 19.992 4077 SER512 C −33.484 −16.93920.958 4078 SER512 O −33.897 −15.859 21.378 4079 SER512 CB −35.369−18.565 20.78 4080 SER512 OG −34.785 −19.668 21.473 4081 CYS513 N−32.281 −17.495 21.329 4082 CYS513 CA −31.384 −16.86 22.278 4083 CYS513C −30.615 −15.692 21.628 4084 CYS513 O −29.947 −14.898 22.298 4085CYS513 CB −30.354 −17.852 22.818 4086 CYS513 SG −31.115 −19.286 23.6434087 GLY514 N −30.674 −15.588 20.256 4088 GLY514 CA −29.821 −14.69519.484 4089 GLY514 C −30.244 −13.23 19.509 4090 GLY514 O −30.297 −12.54718.49 4091 HIS515 N −30.4 −12.719 20.783 4092 HIS515 CA −31.066 −11.43821.038 4093 HIS515 C −30.372 −10.761 22.233 4094 HIS515 O −30.942 −9.97822.991 4095 HIS515 CB −32.567 −11.643 21.32 4096 HIS515 CG −33.314−12.194 20.15 4097 HIS515 ND1 −33.83 −13.466 20.107 4098 HIS515 CD2−33.647 −11.648 18.927 4099 HIS515 CE1 −34.429 −13.627 18.887 4100HIS515 NE2 −34.34 −12.543 18.154 4101 THR516 N −29.009 −10.996 22.3054102 THR516 CA −28.221 −10.644 23.488 4103 THR516 C −26.824 −10.16723.058 4104 THR516 O −26.179 −10.748 22.183 4105 THR516 CB −28.127−11.847 24.46 4106 THR516 OG1 −27.657 −11.48 25.761 4107 THR516 CG2−27.272 −12.999 23.942 4108 ARG517 N −26.341 −9.092 23.773 4109 ARG517CA −24.985 −8.515 23.652 4110 ARG517 C −24.85 −7.641 24.913 4111 ARG517O −25.456 −6.566 24.999 4112 ARG517 CB −24.819 −7.564 22.45 4113 ARG517CG −24.775 −8.14 21.035 4114 ARG517 CD −23.657 −9.149 20.771 4115 ARG517NE −24.174 −10.51 20.9 4116 ARG517 CZ −23.72 −11.583 20.231 4117 ARG517NH1 −24.407 −12.739 20.281 4118 ARG517 NH2 −22.586 −11.579 19.529 4119ALA518 N −24.215 −8.205 25.999 4120 ALA518 CA −24.204 −7.553 27.314 4121ALA518 C −25.608 −7.517 27.958 4122 ALA518 O −25.869 −8.139 28.986 4123ALA518 CB −23.545 −6.175 27.309 4124 GLY519 N −26.512 −6.701 27.309 4125GLY519 CA −27.93 −6.718 27.587 4126 GLY519 C −28.688 −7.409 26.449 4127GLY519 O −28.14 −8.03 25.539 4128 CYS520 N −30.062 −7.253 26.557 4129CYS520 CA −30.985 −7.773 25.539 4130 CYS520 C −31.014 −6.765 24.375 4131CYS520 O −30.996 −5.539 24.549 4132 CYS520 CB −32.391 −7.922 26.13 4133CYS520 SG −33.538 −8.817 25.043 4134 THR521 N −31.107 −7.334 23.121 4135THR521 CA −31.076 −6.541 21.899 4136 THR521 C −31.737 −7.262 20.707 4137THR521 O −32.218 −8.387 20.775 4138 THR521 CB −29.655 −6.04 21.56 4139THR521 OG1 −29.725 −5.151 20.433 4140 THR521 CG2 −28.67 −7.165 21.2684141 LEU522 N −31.81 −6.461 19.576 4142 LEU522 CA −32.287 −6.938 18.2744143 LEU522 C −33.808 −7.211 18.26 4144 LEU522 O −34.349 −7.886 17.3884145 LEU522 CB −31.478 −8.115 17.702 4146 LEU522 CG −30 −7.79 17.4014147 LEU522 CD1 −29.235 −9.081 17.093 4148 LEU522 CD2 −29.848 −6.81216.235 4149 ILE523 N −34.516 −6.48 19.199 4150 ILE523 CA −35.973 −6.53519.323 4151 ILE523 C −36.545 −5.152 19.708 4152 ILE523 O −37.73 −4.98720 4153 ILE523 CB −36.474 −7.617 20.317 4154 ILE523 CG1 −35.954 −7.38121.752 4155 ILE523 CG2 −36.139 −9.03 19.829 4156 ILE523 CD1 −36.643−8.264 22.785 4157 ARG524 N −35.627 −4.116 19.641 4158 ARG524 CA −36.041−2.731 19.862 4159 ARG524 C −36.617 −2.156 18.555 4160 ARG524 O −36.56−2.757 17.481 4161 ARG524 CB −34.864 −1.887 20.374 4162 ARG524 CG−34.488 −2.249 21.814 4163 ARG524 CD −33.444 −1.292 22.384 4164 ARG524NE −33.203 −1.547 23.815 4165 ARG524 CZ −32.502 −2.592 24.316 4166ARG524 NH1 −32.437 −2.773 25.651 4167 ARG524 NH2 −31.859 −3.466 23.5264168 GLN525 N −37.257 −0.95 18.72 4169 GLN525 CA −37.785 −0.124 17.6264170 GLN525 CB −39.314 −0.234 17.512 4171 GLN525 CG −39.806 −1.60917.056 4172 GLN525 CD −39.88 −2.607 18.192 4173 GLN525 OE1 −40.549−2.433 19.205 4174 GLN525 NE2 −39.152 −3.75 17.988 4175 GLN525 C −37.4681.347 17.904 4176 GLN525 O −36.858 1.67 18.923 4177 GLN525 OXT −37.8842.231 16.996

1-23. (canceled)
 24. An isolated polypeptide comprising a polypeptidesequence selected from the group consisting of: (a) an isolatedpolypeptide comprising amino acids 1 to 697 of SEQ ID NO:56; (b) anisolated polypeptide comprising amino acids 2 to 697 of SEQ ID NO:56;(c) an isolated polypeptide comprising a polypeptide sequence encoded bynucleotides 9 to 2099 of SEQ ID NO:55; and (d) an isolated polypeptidecomprising a polypeptide sequence encoded by nucleotides 12 to 2099 ofSEQ ID NO:55.
 25. The isolated polypeptide of claim 24, wherein saidpolypeptide is (a).
 26. The isolated polypeptide of claim 24, whereinsaid polypeptide is (b).
 27. The isolated polypeptide of claim 24,wherein said polypeptide is (c).
 28. The isolated polypeptide of claim24, wherein said polypeptide is (d).
 29. An isolated polypeptideproduced by a method comprising: (a) culturing an isolated recombinanthost cell comprising a vector that comprises the coding region encodingthe polypeptide of claim 24 under conditions such that the polypeptideof claim 24 is expressed; and (b) recovering said polypeptide.
 30. Theisolated polypeptide of claim 24 further comprising a heterologouspolypeptide sequence.
 31. The isolated polypeptide of claim 28 whereinsaid heterologous polypeptide is the Fc domain of immunoglobulin.
 32. Anisolated polypeptide comprising a polypeptide sequence selected from thegroup consisting of: (a) an isolated polypeptide comprising amino acids1 to 694 of SEQ ID NO:54; (b) an isolated polypeptide comprising aminoacids 2 to 694 of SEQ ID NO:54; (c) an isolated polypeptide comprising apolypeptide sequence encoded by nucleotides 9 to 2090 of SEQ ID NO:53;and (d) an isolated polypeptide comprising a polypeptide sequenceencoded by nucleotides 12 to 2090 of SEQ ID NO:53.
 33. The isolatedpolypeptide of claim 32, wherein said polypeptide is (a).
 34. Theisolated polypeptide of claim 32, wherein said polypeptide is (b). 35.The isolated polypeptide of claim 32, wherein said polypeptide is (c).36. The isolated polypeptide of claim 32,.wherein said polypeptide is(d).
 37. An isolated polypeptide produced by a method comprising: (a)culturing an isolated recombinant host cell comprising a vector thatcomprises the coding region encoding the polypeptide of claim 32 underconditions such that the polypeptide of claim 32 is expressed; and (b)recovering said polypeptide..
 38. The isolated polypeptide of claim 32further comprising a heterologous polypeptide sequence.
 39. The isolatedpolypeptide of claim 38 wherein said heterologous polypeptide is the Fcdomain of immunoglobulin.
 40. An isolated polypeptide comprising thepolypeptide encoded by the CAN-12v1 or the CAN-12v2 cDNA clone containedin ATCC Deposit No: PTA-3434.
 41. An isolated polypeptide comprisingamino acids from position 2 through position 587 of SEQ ID NO:56,wherein said polypeptide has calcium-dependent cysteine proteaseactivity.
 42. An isolated nucleic acid comprising a polynucleotideencoding a polypeptide comprising the sequence of amino acids fromposition 90 through position 697 of SEQ ID NO:56, wherein saidpolypeptide has calcium-dependent cysteine protease activity.
 43. Anisolated polypeptide comprising amino acids from position 2 throughposition 584 of SEQ ID NO:54, wherein said polypeptide hascalcium-dependent cysteine protease activity.
 44. An isolated nucleicacid comprising a polynucleotide encoding a polypeptide comprising thesequence of amino acids from position 90 through position 694 of SEQ IDNO:54, wherein said polypeptide has calcium-dependent cysteine proteaseactivity.
 45. An isolated polypeptide having as least 95.0% identity toamino acids 2 to 697 of SEQ ID NO:56, wherein percent identity iscalculated using a CLUSTALW global sequence alignment using defaultparameters, wherein said polypeptide has calcium-dependent cysteineprotease activity.
 46. An isolated polypeptide having as least 95.0%identity to amino acids 2 to 694 of SEQ ID NO:54, wherein percentidentity is calculated using a CLUSTALW global sequence alignment usingdefault parameters, wherein said polypeptide has calcium-dependentcysteine protease activity.